Sphingoid polyalklamine conjugates, isomers and uses thereof

ABSTRACT

The present application discloses methods for stimulating or enhancing an immune response of a subject to protect against an infection caused by an agent selected from Hepatitis B Virus (HBV), avian influenza virus, the bacterium  Bacillus anthracis  or the bacterium  Streptococcus pneumoniae , the method comprising administering to said subject a combination of sphingoid-polyalkylamine conjugate and a biologically active molecule, the combination being effective to provide said stimulation or enhancement of the immune response. Also disclosed are vaccines comprising a combination of the sphingoid-polyalkylamine conjugate and a biologically active molecule for stimulating or enhancing an immune response of a subject to protect against an infection caused by such an agent. Preferred conjugates are N-palmitoyl D-erythro sphingosyl-1-carbamoyl spermine (C-1 CCS), N-palmitoyl D-erythro sphingosyl-3-carbamoyl spermine (C-3 CCS) and mixtures thereof. Also disclosed are uses of C-3 CCS for various applications.

FIELD OF THE INVENTION

The present invention concerns use of sphingolipids' polyalkylamine conjugates for effective delivery of biologically active materials, in particular, antigenic molecules.

LIST OF PRIOR ART

The following is a list of prior art which is considered to be pertinent for describing the state of the art in the field of the invention.

U.S. Pat. No. 5,334,761: “Cationic lipids”;

US 2001/048939: “Cationic reagents of transfection”;

U.S. Pat. No. 5,659,011: “Agents having high nitrogen content and high cationic charge based on dicyanimide dicyandiamide or guanidine and inorganic ammonium salts”;

U.S. Pat. No. 5,674,908: “Highly packed polycationic ammonium, sulfonium and phosphonium lipids”;

U.S. Pat. No. 6,281,371: “Lipopolyamines, and the preparation and use thereof”;

U.S. Pat. No. 6,075,012: “Reagents for intracellular delivery of macromolecules”;

U.S. Pat. No. 5,783,565: “Cationic amphiphiles containing spermine or spermidine cationic group for intracellular delivery of therapeutic molecules”;

Ilies M A. et al. Expert Opin. Ther. Patents. 11(11):1729-1752 (2001);

Miller A D. Chem. Int. Ed. Eng. 37:1768-1785 (1998);

Nakanichi T. et al. J. Control Release 61:233-240 (1999);

Brunel F. et al. Vaccine 17:2192-2193 (1999);

Guy B. et al. Vaccine 19:1794-1805 (2001);

Lima K M et al. Vaccine 19:3518-3525 (2001).

BACKGROUND OF THE INVENTION

Many natural biological molecules and their analogues, including proteins and polynucleotides, foreign substances and drugs, which are capable of influencing cell function at the sub-cellular or molecular level are preferably incorporated within the cell in order to produce their effect. For these agents the cell membrane presents a selective barrier which is impermeable to them. The complex composition of the cell membrane comprises phospholipids, glycolipids, and cholesterol, as well as intrinsic and extrinsic proteins, and its functions are influenced by cytoplasmic components which include Ca⁺⁺ and other metal ions, anions, ATP, microfilaments, microtubules, enzymes, and Ca⁺⁺-binding proteins, also by the extracellular glycocalyx (proteoglycans, glycose aminoglycans and glycoproteins). Interactions among structural and cytoplasmic cell components and their response to external signals make up transport processes responsible for the membrane selectivity exhibited within and among cell types.

Successful delivery of agents not naturally taken up by cells into cells has also been investigated. The membrane barrier can be overcome by associating agents in complexes with lipid formulations closely resembling the lipid composition of natural cell membranes. These formulations may fuse with the cell membranes on contact, or what is more common, taken up by pinocytosis, endocytosis and/or phagocytosis. In all these processes, the associated substances are delivered into the cells.

Lipid complexes can facilitate intracellular transfers also by overcoming charge repulsions between the cell surface, which in most cases is negatively charged. The lipids of the formulations comprise an amphipathic lipid, such as the phospholipids of cell membranes, and form various layers or aggregates such as micelles or hollow lipid vesicles (liposomes), in aqueous systems. The liposomes can be used to entrap the substance to be delivered within the liposomes; in other applications, the drug molecule of interest can be incorporated into the lipid vesicle as an intrinsic membrane component, rather than entrapped into the hollow aqueous interior, or electrostatically attached to aggregate surface. However, most phospholipids used are either zwiterionic (neutral) or negatively charged.

An advance in the area of intracellular delivery was the discovery that a positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), in the form of liposomes, or small vesicles, could interact spontaneously with DNA to form lipid-DNA complexes which are capable of adsorbing to cell membranes and being taken up by the cells either by fusion or more probably by adsorptive endocytosis, resulting in expression of the transgene [Felgner, P. L. et al. Proc. Natl. Acad. Sci., USA 84:7413-7417 (1987) and U.S. Pat. No. 4,897,355 to Eppstein, D. et al.]. Others have successfully used a DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP) in combination with a phospholipid to form DNA-complexing vesicles. The Lipofectin™ reagent (Bethesda Research Laboratories, Gaithersburg, Md.), an effective agent for the delivery of highly anionic polynucleotides into living tissue culture cells, comprises positively charged liposomes composed of the positively charged lipid DOTMA and a neutral lipid—dioleoyl-phosphatidyl-ethanolamine (DOPE) referred to as a helper lipid. These liposomes interact spontaneously with negatively charged nucleic acids to form complexes, referred to as lipoplexes. When excess of positively charged liposomes over DNA negative charges are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces or introduced into the cells either by adsorptive endocytosis or fuse with the plasma membrane, both processes deliver functional polynucleotide into, for example, tissue culture cells. DOTMA and DOTAP are good examples for monocationic lipids. [Illis et al. 2001, ibid.]

Multivalent cations by themselves (including polyamines, inorganic salts and complexes and dehydrating solvents) have also been shown to facilitate delivery of macromolecules into cells. In particular, multivalent cations provoke the collapse of oligo and polyanions (nucleic acids molecules and some peptide molecules and the like) to compact structural forms, and facilitate the packaging of these polyanions into viruses, their incorporation into liposomes, transfer into cells etc. [Thomas T. J. et al. Biochemistry 38:3821-3830 (1999)]. The smallest natural polycations able to compact DNA are the polyamines spermidine and spermine. By attaching a hydrophobic anchor to these molecules via a linker, a new class of transfection vectors, the polycationic lipids and lipopolymers, has been developed.

Cationic lipids and cationic polymers interact electrostatically with the anionic groups of DNA (or of any other polyanionic macromolecule) forming DNA-lipid complexes (lipoplexes) or DNA-polycation complexes (polyplexes). The formation of the complex is associated with the release of counterions of the lipids or polymer, which is thought to be the thermodynamic driving force for lipoplex and polyplex spontaneous formation. The cationic lipids can be divided into four classes: (i) quaternary ammonium salt lipids (e.g. DOTMA (Lipofectin™) and DOTAP) and phosphonium/arsonium congeners; (ii) lipopolyamines; (iii) cationic lipids bearing both quaternary ammonium and polyamine moieties and (iv) amidinium, guanidinium and heterocyclic salt lipids.

SUMMARY OF THE INVENTION

According to a first of its aspects, the vaccination aspect, the present invention concerns a method for stimulating or enhancing an immune response of a subject to provide protection against an infection, the method comprising administering to said subject a combination of sphingoid-polyalkylamine conjugate and a biologically active molecule, the combination being effective to provide said stimulation or enhancement of the immune response, wherein said infection is caused by an agent selected from hepatitis B virus (HBV), avian influenza virus , the bacterium Bacillus anthracis, or the bacterium Streptococcus pneumoniae.

According to a preferred embodiment, the sphingoid-polyalkylamine conjugate comprises a sphingoid backbone carrying, via a carbamoyl linkage, at least one polyalkylamine chain.

The term sphingoid-polyalkylamine conjugate as used herein denotes chemical conjugation (linkage) between a sphingoid base (herein also referred to by the term “sphingoid backbone”) and at least one polyalkylamine chain. The conjugation between the sphingoid base and the at least one polyalkylamine chain is via a carbamoyl linkage, as further detailed hereinafter.

The sphingoid base/backbone, as used herein, includes, long chain aliphatic aminoalcohols, containing two or three hydroxyl groups, the aliphatic chain may be saturated or unsaturated. One example of an unsaturated sphingoid base is that containing a distinctive trans-double bond in position 4. It is noted that the sphingoid backbone has at least two stereoisomeric centers and in accordance with the invention, the stereoisomeric centers may be in R- or S-configuration, as well as combinations of same. In other words, the sphingoid backbone may have at least the following configurations: D-erythro, L-erythro, L-threo, and D-threo.

The term “enhancing” or “stimulating” as used herein includes increase in cellular response and/or humoral response of the immune system upon infection caused by exposure to an agent selected from hepatitis B virus (HBV), avian influenza virus (AIV), the bacterium Bacillus anthracis, or the bacterium Streptococcus pneumoniae as well as, at times, to exposure to a combination of such agents, the increase in the immune response being as a result of the administration of the sphingoid-polyalkylamine conjugate in combination with a biologically active molecule.

The term biologically active molecule as used herein generally denotes any substance which, when administered in combination with the sphingoid-polyalkylamine conjugate, has a protective effect or a therapeutic effect in a subject's body which may be identified (in vitro or in vivo) by known biochemical parameters. For example, the biologically active molecule may include polynucleotides, oligonucleotides, proteins, peptides and drugs having a biochemical effect within a subject's body. In accordance with the vaccination aspect of the invention, the effect may be an enhancing effect on the immune system of a subject. The biologically active material in this case is preferably an antigenic protein, antigenic peptide, antigenic polypeptide, antigenic carbohydrate, or antigenic glyco-protein. The biologically active molecule preferably has a net negative charge or containing one or more regions or moieties carrying a (local) negative charge, such that under suitable condition it interacts with the net positive charge of the sphingoid-polyalkylamine conjugate.

In the context of the vaccination aspect of the present invention, the biologically active molecule may be one or more of the following:

-   -   (i) when said infection is caused by HBV, said biologically         active molecule is an Hepatitis B antigen;     -   (ii) when said infection is caused by avian influenza virus,         said biologically active molecule is an avian influenza antigen;     -   (iii) when said infection is caused by the bacterium Bacillus         anthracis, said biologically active molecule is an antigen of         said bacterium; and     -   (iv) when said infection is caused by the bacterium         Streptococcus pneumoniae, said biologically active molecule is         an antigen of said bacterium.

The stimulation or enhancement is statistically significant better (p<0.005), showing an increase by at least 20%, relative to that elicited by the same biologically active molecule administered without the conjugate.

The invention also concerns the increase of an immune response in cases when the biologically active molecule administered to the subject, without the conjugate, is substantially ineffective in producing such a response.

The term “hepatitis B antigen” refers to any component that is capable, either by itself, with an adjuvant, or in combination with the sphingoid-polyalkylamine in accordance with the invention, to produce an immune response which may be cellular, humoral or both. The antigen may be the whole virus, attenuated, mutated, deactivated or a dead virus. The antigen may also be virus fragments in particular virus membrane fragments. The antigen may further be a molecule or complex of molecules present in the virus produced by isolation or by various biotechnological synthetic technologies. Examples of such molecules are protein or protein fragment, peptide or peptide fragment, nucleic acid molecule, carbohydrate, glycol-protein or low molecular weight compound.

According to a preferred embodiment of the invention, the antigen a particle comprising a hepatitis B surface antigen (HBsAg, i.e. the S peptide), which may be in combination with the pre-S1 or pre-S2 protein.

The term “avian influenza antigen” refers to any component of the avian influenza virus that is capable, either by itself, with an adjuvant, or in combination with the sphingoid-polyalkylamine in accordance with the invention, to produce an immune response which may be cellular, humoral or both. The antigen may be the whole virus, attenuated, mutated, deactivated or a dead virus. The antigen may also be virus fragments. The antigen may further be a molecule or complex of molecules present in the virus produced by isolation or by various biotechnological synthetic technologies. Examples of such molecules are protein or protein fragment, peptide or peptide fragment, nucleic acid molecule, carbohydrate, glycol-protein or low molecular weight compound.

In accordance with one embodiment of the invention, the avian influenza antigen is an “inactivated purified whole avian influenza virus”. The term “inactivated purified whole avian influenza virus” refers to any member of the type A influenza virus which has been manipulated to be in its inactivated, non-pathogenic form. The avian influenza viruses may be categorized into subtypes according to the antigens of the haemagglutinin (H) and neuraminidase (N) molecules expressed on their surfaces and hitherto, and there are known 16 haemagglutinin subtypes and 9 neuraminidase subtypes of influenza A viruses. In the context of the present invention, the inactivated purified whole avian influenza virus preferably relates to those derived from the highly pathogenic Al viruses comprising the H5 and/or the H7 subtypes, as well as the less pathogenic H9 subtype. For example, the inactivated purified whole avian influenza virus may be that derived from the highly pathogenic H5N1 or H5N2 viruses. Additional proteins that can serve as antigens for influenza vaccination comprise the matrix proteins (e.g. M1 and M2 proteins) and the nuclear proteins (e.g. NP).

In accordance with a further embodiment, the avian influenza virus antigen is a virus clade. As known to those versed in the art, phylogenetic analyses of the H5 HA genes from the 2004 and 2005 diseases outbreaks showed 2 different lineages of HA genes, termed clades 1 and 2. A specific example for an avian influenza virus antigen comprises the H5N1 clades.

In accordance with yet another embodiment, the avian influenza antigen may be a whole virion, a split virion, or a virus sub-unit.

The term “antigen of the bacterium Bacillus anthracis” denotes the whole bacteria attenuated, mutated, deactivated or dead, as well as non-encapsulated strains of the bacterium as well as components of the bacterium elaborated by the bacteria that have antigenic activity.

In accordance with one embodiment, the antigen may be a component of the bacterium's capsule.

In accordance with another embodiment, the antigen is a component of the bacterium's toxin. The term “antigen of a toxin produced from the bacterium Bacillus anthracis” which may be used interchangeably with the term “anthrax antigen” denotes any component of the Bacillus anthracis toxin. The anthrax toxin comprises three distinct proteins: lethal factor (LF), oedema factor (OF), and the protective antigen (PA). The PA is a protein that can insert into the membrane of a host cell to create a hole, or pore, through the membrane. The pore then functions as a portal to allow the other two components to get inside of the host cell. The LF is a type of enzyme classified as a zinc protease which attacks and breaks host proteins into smaller and nonfunctional pieces. Destroying host cell proteins is lethal to the host cell, hence the factor's name. The OF is a toxin; the destruction of the host cells allows this toxin to enter the bloodstream, where it can kill cells of the immune system. Disabling the host's immune response allows the bacteria and the toxin to spread throughout the body. In accordance with a preferred embodiment of the invention, the antigen is the protective antigen (PA) moiety of the anthrax toxin.

The term “antigen of Streptococcus pneumoniae” denotes any component of the bacterium including attenuated or inactivated (e.g. inactivated epizootic strains) forms of the bacterium. The streptococci antigen may be a component isolated from the bacteria, a fragment of any native component, a recombinant product comprising a component of the bacterium, a fragment or variant thereof (such as a recombinant protein, a fused protein, etc.), a modified component (e.g. chemically modified). Preferably, the Streptococci antigen comprises a polysaccharide or protein conjugated antigen derived from the bacterium, the polysaccharide antigen being preferably conjugated to a protein carrier. It is noted that polysaccharide antigens being preferably conjugated to a protein carrier are commonly used for polysaccharide-based vaccines, although, it is to be understood that the invention is not limited to such conjugations. Non-limiting commercial products which may be used in accordance with this embodiment include mixtures of several strains, such as 23-valent polysaccharide vaccine Pneumovax™ (Merck NJ USA), and 7-valent conjugated vaccine (for young children), (PrevNar™ or PCV7 (Wyeth, NJ, USA).

The present invention concerns a vaccine comprising a combination of a sphingoid-polyalkylamine conjugate and an amount of a biologically active molecule, the amount of said biologically active molecule, when combined with said sphingoid-polyalkylamine conjugate, being effective to stimulate or enhance an immune response of a subject against an infection, said infection being caused by an agent selected from one or more of the following: HBV, avian influenza virus, the bacterium Bacillus anthracis, and the bacterium Streptococcus pneumoniae.

According to yet another embodiment, the invention provides a complex comprising a sphingoid-polyalkylamine conjugate and a biologically active molecule, the complex being capable of enhancing or stimulating an immune response of a subject to provide protection against an infection, said infection being caused by an agent selected from one or more of the following: HBV, avian influenza virus, the bacterium Bacillus anthracis, and the bacterium Streptococcus pneumoniae.

In accordance with a second aspect, the present invention relates to a method for the treatment or prevention of a disease or disorder comprising administering to a subject in need of the treatment a combination comprising a biologically active molecule and a sphingoid-polyalkylamine conjugate having the general formula (I′):

wherein

R₁ represents a hydrogen, a branched or linear alkyl, saturated or unsaturated cycloalkyl, aryl, hydroxyalkyl, alkylamine, or a group —C(O)R₅;

R₂ and R₅ represent, independently, a branched or linear C₁₀-C₂₄ alkyl, hydroxyalkyl, alkenyl, saturated or unsaturated cycloalkyl, aryl or polyenyl groups;

R₃ is a group —C(O)—NR₆R₇, R₆ and R₇ represent, independently, a hydrogen, or a saturated or unsaturated branched or linear polyalkylamine, wherein one or more amine units in said polyalkylamine may be a quaternary ammonium;

W represents a group selected from —CH═CH—, —CH₂—CH(OH)— or —CH₂—CH₂—.

A preferred sphingoid-polyalkylamine conjugate of formula (I′) is N-palmitoyl D-erythro sphingosyl-3-carbamoyl spermine (C-3 CCS).

In accordance with this aspect of the invention, there is also provided a pharmaceutical composition comprising a biologically active molecule in combination with said sphinogid-polyalkylamine conjugate of formula (I′). The composition may include other sphinogid-polyalkylamine conjugates, such as those included in general formula (I).

According to one embodiment, the sphinogid-polyalkylamine conjugate of formula (I′) is mixed with at least one additional sphinogid-polyalkylamine conjugate, such as N-palmitoyl D-erythro sphingosyl-1-carbamoyl spermine (C-1 CCS).

Further, according to an embodiment, the sphinogid-polyalkylamine conjugate of formula (I′) is used for the preparation of vaccines.

BRIEF DESCRIPTION OF THE FIGURES

In order to understand the invention and to see how it may be carried out in practice, some embodiments will now be described, by way of non-limiting examples only, with reference to the accompanying figures, in which:

FIGS. 1A-1E show several possible chemical structures, “linear”, “branched” or “cyclic” cationic lipid compounds which are encompass under the general definition of sphingoid-polyalkylamine conjugate of formula (I), wherein FIGS. 1A-1B shows a sphingoid backbone (ceramide) linked to a single polyalkylamine chain, FIG. 1C and FIG. 1D show the same sphingoid backbone linked to two polyalkylamine chains, FIG. 1E shows again the same backbone, however, in which a single polyalkylamine chain is linked via the two hydroxyl moieties to form a cyclic polyalkylamine conjugate.

FIGS. 2A-2F show the bio-distribution and pharmacokinetics of various fluorescently-labeled lipid formulations (liposomes), with and without the influenza antigens (referred to as HN) following intranasal (i.n.) administration to BALB/C mice: in the nose -▾-, GI-

-, lungs -♦- or spleen — with unrecovered -

-: FIG. 2A shows distribution of empty DMPC:DMPG (mole ratio 9:1); FIG. 2B shows distribution of empty DOTAP:cholesterol; FIG. 2C shows distribution of empty N-palmitoyl D-erythro sphingosyl carbamoyl spermine (CCS):cholesterol; FIG. 2D shows distribution of DMPC:DMPG:HN; FIG. 2E shows distribution of DOTAP:cholesterol:HN; and FIG. 2F shows distribution of CCS:cholesterol:HN,

FIGS. 3A-3D show bio-distribution of various ¹²⁵I-HN loaded liposome formulations, administered as above, in the nose -▾-, GI-

-, lungs -▾- or spleen -

- with unrecovered

, and in particular, FIG. 3A shows bio-distribution of free ¹²⁵I-HN; FIG. 3B shows ¹²⁵I-HN loaded liposomes composed of DOTAP:Cholesterol; FIG. 3C shows ¹²⁵I-HN loaded liposomes composed of DMPC:DMPG and FIG. 3D shows ¹²⁵I-HN loaded liposomes composed of CCS:Cholesterol.

FIG. 4 shows mean serum HI (haemagglutination inhibition) antibody titers against A/New Caledonia virus following vaccination of rats i.m., once, with 12 μg total HA (trivalent vaccine), with or without CCS/C (mole ratio 3:2), at a lipid:antigen w/w ratio of 150:1, as determined by the HI assay.

FIG. 5 shows the mean sum of viral titer (log₁₀TCID₅₀/ml) in nasal wash following i.m. vaccination of ferrets with free HA (F-HA) or with CCS/C-HA and challenge with the homologous influenza A/New Caledonia virus (H1N1) on day 28. P<0.001 as compared to CCS/C only and F-HA.

FIG. 6 shows the hemagglutination inhibition (HI) antibody levels in serum of BALB/C mice at weeks 2, 4, 8, 12 and 20 after a single intramuscular (i.m.) immunization with avian influenza (H5N1) vaccine at doses of 3 μg or 6 μg HA.

FIGS. 7A-7B shows serum anti-HBsAg antibody levels 5 and 12 weeks following intraperitoneal (i.p.) or intranasal (i.n.) vaccination (FIG. 7A) and the specific isotypes against HBsAg detected by ELISA 6 weeks post-vaccination (FIG. 7B) of BALB/C mice with the free or liposomal (CCS/C) HBV vaccine.

FIG. 8A-8C show Anti-HBsAg (FIG. 8A), anti-HBsAg IgG1 (FIG. 8B) and anti-HBsAg IgG2a (FIG. 6C) titers in serum collected 14, 28 days, 2 months and 3 months following a single intraperitoneal (i.p.) vaccination of BALB/C mice with free antigen (F-Ag), the commercial vaccine Sci-B-Vac™ (Alum adsorbed Ag) or the liposomal antigen (CCS/C-Ag).

FIG. 9A-9B are bar graphs showing serum anti-B. anthracis PA IgG antibodies median levels in samples from BALB/C mice following subcutaneous (s.c) or intranasal (i.n.) vaccination (a single or double-dose vaccination, respectively) with free antigen (F-Ag), Alum-Ag at two w/w ratios (7/1, 25/1), or liposomal CCS/Cholesterol-antigen (CCS/C-Ag). FIG. 9A shows the O.D (presenting levels) 2 and 4 weeks post-vaccination as determined using a commercial kit: QuickELISA™ Anthrax-PA kit (Immunetics, Inc, MA, USA) (% indicates percentage of responders. OD>0.186 was considered a positive response). FIG. 9B shows the titers as determined by ELISA (as described herein below) where only i.n. vaccination was tested also after 7 weeks (2 weeks post the second vaccination), (%: percentage of responders (titer>30)).

FIG. 10 is a bar graph showing anti-PA antibodies median levels in serum samples of Guinea pigs 28 days after a single subcutaneous vaccination with free antigen (F-PA), liposomal CCS/Cholesterol antigen (CCS/C-PA) or Sterne strain (STI, commercial veterinary vaccine used as a positive control); as determined by a commercial kit: QuickELISA™ Anthrax-PA kit (Immunetics, Inc, MA, USA). (% indicates percentage of responders. OD>0.186 was considered a positive response).

DETAILED DESCRIPTION

In accordance with a first of its aspects, the present invention concerns the use of sphingoid-polyalkylamine conjugates as adjuvants for biologically active molecules (e.g. antigens) for enhancing the immune response of a subject, for the purpose of treating the subject against an infection caused by an agent selected from the group consisting of hepatitis B virus (HBV), avian influenza virus (AIV), the bacterium Bacillus anthracis and the bacterium Streptococcus pneumoniae.

The sphingoid-polyalkylamine conjugates are cationic lipids compounds, which may be synthesized in the following manner. N-substituted long-chain bases in particular, N-substituted sphingoids or sphingoid bases are coupled together with different polyalkylamines or their derivatives, to form a polyalkylamine-sphingoid entity, which is used as is, or further alkylated. Some sphingoid-polyalkylamine conjugates are also commercially available.

Protonation at a suitable pH or alkylation of the formed polyalkylamine-sphingoid entity attributes to the lipid-like compounds a desired positive charge for interaction with biologically active biological molecules to be delivered into target cells and with the targeted cells. The sphingoid-polyalkylamine conjugates may be efficiently associated with the biologically active molecules by virtue of electrostatic interactions between the anionic character of the biologically active molecules and the polyalkylamine moieties of the conjugate to form complexes (lipoplexes).

Thus, disclosed herein is a method for stimulating or enhancing an immune response of a subject to provide protection against an infection, the method comprising administering to said subject a combination of at least one sphingoid-polyalkylamine conjugate and a biologically active molecule, the combination being effective to provide said stimulation or enhancement of the immune response, wherein said infection is caused by an agent selected from the group consisting of hepatitis B virus (HBV), avian influenza virus (AIV), the bacterium Bacillus anthracis and the bacterium Streptococcus pneumoniae, wherein said sphingoid-polyalkylamine conjugate comprises a sphingoid backbone carrying, via a carbamoyl linkage, one or two polyalkylamine chains.

In accordance with a preferred embodiment, the sphingoid-polyalkylamine conjugate includes a linkage between a sphingoid backbone and at least one polyalkylamine chain, the linkage is via corresponding carbamoyl linkage.

More preferably, the sphingoid-polyalkylamine conjugate has the general formula (I):

wherein

R₁ represents a hydrogen, a branched or linear alkyl, saturated or unsaturated cycloalkyl, aryl, hydroxyalkyl, alkylamine, or a group —C(O)R₅;

R₂ and R₅ represent, independently, a branched or linear C₁₀-C₂₄ alkyl, hydroxyalkyl, alkenyl, saturated or unsaturated cycloalkyl, aryl or polyenyl groups;

R₃ and R₄ are independently a hydrogen or a group —C(O)—NR₆ R₇, R₆ and R₇ being the same or different for R₃ and R₄ and represent, independently, a hydrogen, or a saturated or unsaturated branched or linear polyalkylamine, wherein one or more amine units in said polyalkylamine may be a quaternary ammonium; wherein at least one of said R₃ or R₄ are not simultaneously a hydrogen; or

R₃ and R₄ form together with the oxygen atoms to which they are bound a heterocyclic ring comprising —C(O)—NR₉—[R₈—NR₉]_(m)—C(O)—, R₈ represents a saturated or unsaturated C₁-C₄ alkyl and R₉ represents a hydrogen or a polyalkylamine of the formula —[R₈—NR₉]_(n)—, wherein said R₉ or each alkylamine unit R₈NR₉ may be the same or different in said polyalkylamine; and

n and m are independently an integer from 1 to 10, preferably 3 to 6;

W represents a group selected from —CH═CH—, —CH₂—CH(OH)— or —CH₂—CH₂—.

According to one preferred embodiment the sphingoid backbone is a ceramide having a carbamoyl moiety linked to one (FIG. 1A-1B) or two (FIGS. 1C-1D) polyalkylamine chains, or linked via two carbamoyl moieties to a single polyalkylamine chain, to form a cyclic polyalkylamine conjugate (FIG. 1E).

When referring to the use of the sphingoid-polyalkylamine conjugate for vaccination, the biologically active material is any molecule which when administered with the sphingoid-polyalkylamine conjugate has an effect on the immune system of a subject, according to one embodiment, a stimulating or enhancing effect as compared to the effect of the biologically active material when provided without the conjugate. The effect is preferably by a factor of two or more relative to the effect, if any, of the biologically active molecule, when provided to a subject without said conjugate. Thus, the sphingoid-polyalkylamine conjugate is to be considered an efficacious adjuvant for vaccination.

According to one embodiment, the biologically active material is a protein, polypeptide, peptide, glycol-protein or carbohydrate, being derived from (either by isolation, modification and/or production) HBV, avian influenza virus, the bacterium Bacillus anthracis, or the bacterium Streptococcus pneumoniae. Specifically, the biologically active molecule may be antigenic protein, antigenic peptide, antigenic carbohydrate, antigenic glycol-protein and immunostimulants. Antigenic proteins and peptides and immunostimulants are all well known in the art. Preferably, the biologically active protein or peptide or carbohydrate has at a physiological pH either a net negative dipole moment, a net negative charge or contains at least one negatively charged region.

Preferred sphingoid-polyalkylamine conjugates according to this aspect of the invention comprise the different structural and stereoisomers of N-palmitoyl D-erythro sphingosyl carbamoyl-spermine (CCS). This conjugate includes a ceramide linked via a C-1 and/or C-3 carbamoyl linkage to spermine as well as to mixtures of same. Further preferred sphingoid-polyalkylamine conjugates according to the invention comprise N-palmitoyl D-erythro sphingosyl-1-carbamoyl spermine (C-1 CCS), N-palmitoyl D-erythro sphingosyl-3-carbamoyl spermine (C-3 CCS) as well as mixtures of same and the various stereoisomers of same. In the following text, the abbreviation CCS denotes either a single conjugate or a mixture of same, unless otherwise specifically stated.

According to one embodiment, the sphingoid-polyalkylamine conjugate, and preferably the CCS, is used for the preparation of a vaccine for hepatitis B virus (HBV). To this end, the conjugate is formulated with a HBV antigen to form a vaccine (which may include other constituents). A specifically preferred HBV derived antigenic material is the HBsAg particle.

According to another embodiment, the sphingoid-polyalkylamine conjugate, and preferably the CCS, is used for the preparation of a vaccine for avian influenza virus (AIV). In this particular embodiment, the biologically active material is preferably an inactivated purified whole avian influenza virus or a component thereof comprising at least one or a combination of the haemagglutinin (H or HA) and neuraminidase (N or NA) antigens (the combination referred to as HN, unless otherwise stated), or a biologically active analog of a molecule derived from these antigens, which is capable of eliciting an immune response against the natural antigen(s) as defined hereinabove. A preferred inactivated purified whole avian influenza virus in accordance with the invention is the whole virus comprising the H5N1 antigens.

According to yet another embodiment, the sphingoid-polyalkylamine conjugate, and preferably the CCS, is used for the preparation of a vaccine for the bacterium Bacillus anthracis (Anthrax). In this particular embodiment, the biologically active material is preferably an antigen of a toxin produced from the bacterium Bacillus anthracis or the immunogenic component thereof. In accordance with a preferred embodiment, the antigen comprises one or more of the bacterium toxin proteins selected from the lethal factor (LF), oedema factor (OF), and the protective antigen (PA), or a biologically active analog of the protein, which is capable of eliciting an immune response against the bacterium. A preferred antigen in accordance with this embodiment of the invention is the protective antigen.

In accordance with yet a further embodiment, the sphingoid-polyalkylamine conjugate, and preferably the CCS, is used for the preparation of a vaccine for the bacterium Streptococcus pneumoniae. In accordance with a preferred embodiment, the vaccine comprises as the biologically active molecule an inactivated or attenuated bacterium, an antigen, or combinations of antigens derived from the Streptococcus pneumoniae bacterium.

Various Streptococcus pneumoniae antigens have already been described in the art and the following is a non-limiting description of some such antigens which may be utilized in accordance some embodiments of the invention. Needless to say that in the context of the present invention, one or more antigens derived from Streptococcus pneumoniae may be utilized.

In accordance with one embodiment, there is disclosed the use of a recombinant CIpP protein derived from Streptococcus pneumoniae as an antigen. CIpP protein of Streptococcus pneumoniae is serine protease having 21 kDa of molecular weight (Genebank AE008443) which is one of heat shock proteins. Recombinant CIpP protein can be prepared by large scale expression and isolation in accordance with conventional genetic engineering techniques in the art.

In accordance with a further embodiment, the antigen may be a capsular saccharide antigen, which is preferably conjugated to a carrier protein. The saccharide antigen may include mixtures of polysaccharides from different serotypes (e.g. from the 90 different serotypes) from which there are 23 serotypes widely used (1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33). As known to those versed in the art, PrevNar™, a commonly known vaccine, contains antigens from seven serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) with each saccharide individually conjugated to diphtheria CRM₁₉₇ protein (a nontoxic variant of diphtheria toxin) isolated from cultures of Corynebacterium diphtheriae strain C7 (β197) by reductive amination. Pneumovax™ is another polysaccharide-based vaccine containing polysaccharides from the above mentioned 23 different strains.

In accordance with yet another embodiment, the antigen may be a Streptococcus pneumoniae protein. Non-limiting examples of Streptococcus pneumoniae proteins which may be used as Streptococcus pneumoniae antigens, alone or in combination, e.g. with a Streptococcus pneumoniae saccharide include Poly Histidine Triad family (Pht; e.g. PhtA, PhtB, PhtD, or PhtE), Lyt family (e.g. LytA, LytB, or LytC), SpsA, Sp128, Sp130, Sp125, Sp101 and Sp133, or truncate or immunologically functional equivalent thereof, optionally with a Th1 adjuvant (an adjuvant inducing a predominantly Th1 immune response). Specific, however, non-limiting Streptococcus pneumoniae proteins include pneumococcal surface protein A (PspA [Nabors et al. Vaccine 18:1743-1754 (2000), incorporated herein in its entirety, by reference]), pneumococcal surface protein C (PspC), pneumococcal surface immunogenic protein B (PsipB), pneumococcal adherence and virulence factor A (PavA), pneumococcal glutamyl-tRNA synthetase, pneumolysin, cholin binding protein A (CbpA), pneumococcal autolysin (LytA, mentioned above) as well as pneumococcal surface adhesion A (PsaA) which is a streptococcal common protein (Russell et al., J. Clin. Microbiol., 28:2191-2195 (1990); and U.S. Pat. No. 5,422,427). The antigen may also be any peptide comprising the epitope of the above mentioned proteins. The complete genome sequence of a virulent isolate of Streptococcus pneumoniae was determined and several surface exposed proteins that may serve as potential vaccine candidates were identified [Tettelin et al. Science 293:498-506 (2001); and Hoskins et al. J Bacteriology, 183(19):5709-5717 (2001), both incorporated herein in their entirety, by reference].

In a further embodiment, the antigen may comprise a phosphocholine, which is an antigenic component (of the bacterial cell wall) in a variety of pathogenic organisms.

Further, the antigen may be a Streptococcus pneumoniae derived pili or a pilus subunit or subunits. Examples of three pilus subunits are RrgA, RrgB and RrgC as described by P. Ruggiero et al. [Infection and Immunity 75(2):1059-1062 (2007)]

The sphingoid-polyalkylamine conjugate may form part of a kit for the preparation of a pharmaceutical composition for stimulating or enhancing the immune response of a subject when combined with a biologically active material. Specifically, the kit may be for the preparation of a pharmaceutical composition for stimulating or enhancing an immune response of a subject so as to provide the same with protection against an infection caused by an agent selected from hepatitis B virus (HBV), avian influenza virus (AIV), the bacterium Bacillus anthracis and the bacterium. Streptococcus pneumoniae. The kit comprises a combination of sphingoid-polyalkylamine conjugate and the biologically active material, the biologically active material being selected such that:

-   -   (i) when the infection is caused by HBV, said biologically         active material is an Hepatitis B antigen;     -   (ii) when the infection is caused by avian influenza virus, said         biologically active molecule comprises an avian influenza         antigen;     -   (iii) when the infection is caused by the bacterium Bacillus         anthracis, said biologically active molecule comprises an         antigen of said bacterium;     -   (iv) when the infection is caused by the bacterium Streptococcus         pneumoniae, said biologically active molecule comprises an         antigen of said bacterium.

The kit may comprise, in addition to said conjugate, instructions for use of same in combination with one or more of the above defined biologically active materials. The conjugate in the kit may be in a dry form, in which case, the kit may also include a suitable fluid with which the conjugate is mixed prior to use to form a suspension, emulsion or solution, or it may already be in a fluid (suspension, emulsion, solution, etc.) form.

Disclosed herein is also a complex comprising the sphingoid-polyalkylamine conjugate as defined herein and preferably N-palmitoyl-D-erythro-sphingosyl carbamoyl-spermine (CCS), being a single isomer or mixture of CCS isomers, and the biologically active molecule, the complex being capable of enhancing or stimulating an immune response of a subject to provide protection against an infection caused by an agent selected from HBV, AIV, the bacterium Bacillus anthracis or the bacterium Streptococcus pneumoniae. The biologically active molecule is as defined hereinabove. The complex is preferably forms a lipid assembly, and the lipid assembly is preferably a liposome, a vesicle or a combination of same.

In accordance with a further aspect there is provided a method for the treatment or prevention of a disease or disorder comprising administering to a subject in need of the treatment a composition comprising a biologically active molecule and a sphingoid-polyalkylamine conjugate having the general formula (I′):

wherein

R₁ represents a hydrogen, a branched or linear alkyl, saturated or unsaturated cycloalkyl, aryl, hydroxyalkyl, alkylamine, or a group —C(O)R₅;

R₂ and R₅ represent, independently, a branched or linear C₁₀-C₂₄ alkyl, hydroxyalkyl, alkenyl, saturated or unsaturated cycloalkyl, aryl or polyenyl groups;

R₃ is a group —C(O)—NR₆ R₇, R₆ and R₇ represent, independently, a hydrogen, or a saturated or unsaturated branched or linear polyalkylamine, wherein one or more amine units in said polyalkylamine may be a quaternary ammonium;

W represents a group selected from —CH═CH—, —CH₂—CH(OH)— or —CH₂—CH₂—.

This aspect is at times referred to as the “C-3 conjugate aspect” of the invention.

In accordance with the C-3 conjugate aspect of the invention, the method is applicable for the treatment or prevention of any disease or disorder, where the conjugate elicits or stimulates the therapeutic effect of the biologically active molecule. In accordance with one embodiment, the method is for stimulating or enhancing an immune response of a subject to provide protection against an infection.

The invention also provides pharmaceutical compositions comprising at least the sphingoid-polyalkylamine conjugate of formula (I′) in combination with a biologically active molecule.

The conjugate of general formula (I′) is preferably N-palmitoyl D-erythro sphingosyl-3-carbamoyl spermine.

The sphinogid-polyalkylamine conjugate of formula (I′) may be used as the only sphinogid-polyalkylamine conjugate or in combination with other sphinogid-polyalkylamine conjugates such as those defined by formula (I).

One preferred combination comprises, without being limited thereto, a sphinogid-polyalkylamine conjugate mixture comprising a first sphingoid-polyalkylamine conjugate being N-palmitoyl-D-erythro-sphingosyl-3-carbamoyl spermine and a second sphingoid-polyalkylamine conjugate being N-palmitoyl-D-erythro-sphingosyl-1-carbamoyl spermine.

When using a mixture of sphinogid-polyalkylamine conjugates, the mole:mole ratio between a first sphingoid-polyalkylamine conjugate of general formula (I′) and a second sphingoid-polyalkylamine conjugate of formula (I) may be any ratio as desired.

The conjugate of formula (I) or of formula (I′) preferably comprises one or more of the following:

R₁ preferably represents a —C(O)R₅ group;

R₂ and R₅ preferably represent, independently, a linear or branched C₁₂-C₁₈ alkyl or alkenyl groups;

W preferably represents —CH═CH—;

In accordance with one preferred embodiment, the sphinogid-polyalkylamine conjugate of formula (I) or (I′) comprises a polyalkylamine having the general formula (II):

wherein

R₈ represent a C₁-C₄ alkyl, preferably C₃-C₄ alkyl;

R₉ represents a hydrogen or a polyalkylamine branch of formula (II), said R₈ and R₉ may be the same or different for each alkylamine unit, —R₈NR₉—, in the polyalkylamine of formula (II); and

n represents an integer from 3 to 6.

A preferred group of polyalkylamine chains forming part of the sphingoid-polyalkylamine conjugates comprises spermine, spermidine, a polyalkylamine analog or a combination of same thereof. The term polyalkylamine analog is used to denote any polyalkylamine chain, and according to one embodiment denotes a polyalkylamine comprising 1 to 10 amine groups, preferably from 3 to 6 and more preferably 3 or 4 amine groups. Each alkylamine within the polyalkylamine chain may be the same or different and may be a primary, secondary, tertiary amine or quaternary ammonium.

The alkyl moiety, which may be the same or different within the polyalkylamine chain, is preferably a C₁-C₆ aliphatic repeating unit. Some non-limiting examples of polyalkylamines include spermidine, N-(2-aminoethyl)-1,3-propane-diamine, 3,3′-iminobispropylamine, spermine and bis(ethyl) derivatives of spermine, polyethyleneimine.

The sphingoid-polyalkylamine conjugates may be further reacted with methylation agents in order to form quaternary ammonium salts. The resulting compounds are positively charged to a different degree depending on the ratio between the quaternary, primary and/or secondary amines within the formed conjugates. As such, the sphingoid-polyalkylamine conjugate exists as quaternized nitrogen salt including, but not limited to, quaternary ammonium chloride, a quaternary ammonium iodide, a quaternary ammonium fluoride, a quaternary ammonium bromide, a quaternary ammonium oxyanion and a combination thereof.

Non-limiting examples of the sphingoids or sphingoid bases which may be used according to a more specific embodiment of the invention, include, sphingosines, dihydrosphingosines, phytosphingosines, dehydrophytosphingosine and derivatives thereof. Non-limiting examples of such derivatives are acyl derivatives, such as ceramide (N-acylsphingosine), dihydroceramides, phytoceramides and dihydrophytoceramides, respectively, as well as ceramines (N-alkylsphinogsine) and the corresponding derivatives (e.g. dihydroceramine, phytoceramine etc.). The suitably N-substituted sphingoids or sphingoid bases posses free hydroxyl groups which are activated and subsequently reacted with the polyalkylamines to form the polyalkylamine-sphingoid entity. Non-limiting examples of activation agents are N,N′-disuccinimidylcarbonate, di- or tri-phosgene or imidazole derivatives. The reaction of these activation agents with the sphingoids or the sphingoid bases yields a succinimidyloxycarbonyl, chloroformate or imidazole carbamate, respectively, at one or both hydroxyls. The reaction of the activated sphingoids with polyalkylamines may yield mono-substituted, di-substituted, branched, straight (unbranched) or cyclic conjugates.

When referring to all aspects of the invention, the sphingoid-polyalkylamine conjugate (of formula (I) and/or of formula (I′)) may be in the form of free conjugate or as part of a lipid assembly. One example of a suitable lipid assembly comprises micelles or vesicles (liposomes). Other examples of assemblies include the formation of micelles, inverted phases, cubic phases and the like. Evidently, the sphingoid polyalkylamine conjugate may be in combined vesicle/micelle form or any other combination of assemblies. The assemblies may be loaded with the biologically active molecules. It is noted that CCS, either the isolated isomer, the mixture of isolated isomers, or the synthesized mixture of CCS, do not form liposomes by themselves, i.e. they are not considered as liposome-forming lipids and thus require a helper lipid (such as cholesterol or DOPE). Lipid assemblies in the context of the present invention comprise CCS in combination with at least one helper lipid.

Lipid assembly as used herein denotes an organized collection of lipid molecules forming inter alia, micelles, liposomes and other forms of lipid assemblies. The lipid assemblies are preferably stable lipid assemblies. Stable lipid assembly as used herein denotes an assembly being chemically and physically stable under storage conditions (4° C., in physiological medium) for at least one day, preferably one week and more preferably more than a month.

When the assemblies are in the form of vesicles (e.g. liposomes), the biologically active molecule may be encapsulated within the vesicle, form part of its lipid bilayer, or be adsorbed to the surface of the vesicle (or any combination of these three options). When the assemblies are micelles, the biologically active molecules may be inserted into the amphiphiles forming the micelles and/or associated with it electrostatically, in a stable way.

According to one embodiment, the assemblies form liposomes. The formed liposomes may be shaped as unsized heterogeneous and heterolamellar vesicles (UHV) having a diameter of about 50-5000 nm. The formed UHV may be downsized and converted to (more homogenous) unilamellar vesicles having a diameter of about 50-100 nm by further processing. The structure and dimensions of the vesicles, e.g. their shape, lamellarity and size, may have important implications on their efficiency as vehicles for delivery of the active biological entities to the target, i.e. these determine their delivery properties. It is noted that the unilameller liposomes may be either small vesicles (equal or less than 50 nm in diameter) as well as large unilamellar vesicles.

Thus, as used herein, the terms “encapsulated in”, “contained in”, “loaded onto” or “associated with” indicate a physical association between the lipid conjugate and the biologically active molecule. The physical association may be either containment or entrapment of the molecule within assemblies (e.g. vesicles, micelles or other assemblies) formed from the conjugate; non-covalent linkage of the biological molecule to the surface of such assemblies, or embedment of the biological molecule in between the sphingoid-polyalkylamine conjugates forming such assemblies. Without being bound by theory, it is believed that association of the sphingoid-polyalkylamine conjugate to the biological molecule, under physiological conditions is related to the positive charge or positive dipole of the conjugate.

The invention should not be limited by the particular type of association formed between the sphingoid-polyalkylamine conjugate and the biologically active molecule. Thus, association means any interaction between the conjugate or the assembly formed therefrom and the biologically active material which is capable of achieving a desired therapeutic (as well as prophylactic) effect.

The biologically active molecule and the conjugate may be combined by any method known in the art. This includes, without being limited thereto, post- or co-lyophilization of the conjugate with the biologically active molecule, or by mere mixing of preformed sphingoid-polyalkylamine conjugate with the biological molecule. Method for co-lyophilization are described, inter alia, in U.S. Pat. Nos. 6,156,337 and 6,066,331, while methods for post-encapsulation are described, inter alia, in WO03/000227, all incorporated herein by reference.

A preferred weight ratio between the sphingoid-polyalkylamine conjugate and biologically active material is 1000:1 to 1:1 weight ratio, with preferably 300:1 to 10:1 weight ratio. It is noted that the biological activity of the composition comprising the conjugate and the biologically active material does not necessarily have to be linear with the increase in said ratio and at times the “vaccination behavior” may have a bell shape profile.

The sphingoid-polyalkylamine conjugate may be used as a sole adjuvant or in combination with additional adjuvants, so as to provide an increase in response to the biologically active molecule such as antigenic molecules. Such substances include, for example, immunostimulating agents (i.e. “adjuvants”), which when added to a vaccine they improve the immune response to the antigen so that a lower antigen dose can be used while producing a greater response as compared to that without the CCS or other adjuvants. The immunostimulating agent may be delivered together with the conjugate/biologically active material, or within a specified time interval (e.g. several hours or days before or after the administration of the conjugate/biologically active molecule).

Preferred immunostimulating agents include, without being limited thereto, cytokines, such as interleukins (IL-2, IL-12, IL-15, IL-18), interferons (IFN alpha, beta, gamma), GM-CSF, synthetic oligodeoxynucleotides (ODN, e.g. CpG), bacterial toxins (e.g. cholera toxin [CT], staphylococcal enterotoxin B [SEB], heat labile E. Coli enterotoxin [HLT]), as well as any other adjuvants known to be used in the art for enhancing or stimulating the immune response to an antigenic molecule.

Other pharmaceutically acceptable adjuvants which may be used in combination with the sphingoid-polyalkylamine conjugate and the biologically active material include, without being limited thereto, aluminum hydroxide, alum, QS-21, monophosphoryl lipid A, and 3-O-deacylated monophosphoryl lipid A (3D-MPL).

It is noted that the sphingoid-polyalkylamine conjugates of the invention exhibited an activity of an adjuvant per se. The results presented herein show that the sphingoid-polyalkylamine conjugate of the invention and in particular, N-palmitoyl D-erythro sphingosyl carbamoyl-spermine (including the C-1 and C-3 isomers of CCS and their synthetic mixtures) which are discussed below, may act as efficacious and safe adjuvants for a broad spectrum of active substances, such as antigens. For example, it has been shown that vaccines comprising an influenza antigens or hepatitis B surface antigen (HBsAg), in combination with a single CCS isomer (e.g. C-1 or C-3 isomer), or a mixture of isomers, induced a faster, stronger and more durable immune response in mice, as compared to these commercial vaccines administered without an adjuvant or combined with other adjuvants, such as alum. CCS may thus allow reduction of the dose and number and frequency of administrations of a biologically active molecule, such as an antigen.

The assemblies may include in addition to the sphingoid-polyalkylamine conjugate (non-methylated or methylated) other helper lipid substances. Such helper lipid substances may include non-cationic lipids like DOPE, DOPC, DMPC, Cholesterol, oleic acid or others at different mole ratios to the lipid-like compound. Cholesterol is one preferred added substance for in vivo application while DOPE may be a preferred helper lipid for in vitro applications. In this particular embodiment the mole ratio of cholesterol to the cationic lipid is within the range of 0.01-1.0 and preferably 0.1-0.4.

The assemblies may also include enhancers and co-factors (as known in the art, such as CaCl₂) and soluble polyalkylamines.

Other components which may be included in the lipid assembly, and which are known to be used in structures of the like, are steric stabilizers. One example of a commonly used steric stabilizer is the family of lipopolymers, e.g. polyethylene glycol derivatized lipids (PEG-lipid conjugate). This family of compounds are known, inter alia, to increase (extend) the blood circulation time of lipids.

The methods disclosed herein comprises administration of the sphingoid-polyalkylamine conjugate and biologically active material either together, or within a predefined time interval, such as several hours or several days (optionally in combination with an immunostimulant). However, according to one embodiment, the conjugate and biologically active material are mixed together prior to administration to the subject.

Administration of the sphingoid-polyalkylamine conjugate together with the biologically active material concerns another aspect of the invention. Accordingly, there is provided a pharmaceutical composition comprising a physiologically acceptable carrier and an effective amount of the sphingoid-polyalkylamine conjugate together with the biologically active material. The sphingoid-polyalkylamine conjugate may be incorporated in the composition as part of a carrier, as an adjuvant, and may be combined with other pharmaceutically acceptable adjuvants, such as immunostimulants.

The sphingoid-polyalkylamine conjugated in combination with the biologically active material may be administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual subject, the site and method of administration, scheduling of administration, subject age, sex, body weight and other factors known to medical practitioners.

The “effective amount” for purposes herein denotes an amount which is effective to achieve the desired therapeutic or prophylactic effect. The effective amount is typically determined in appropriately designed clinical trials (dose range studies) and the person versed in the art will know how to properly conduct such trials in order to determine the effective amount.

When referring to vaccination, the amount must be effective to exhibit an enhancement or induction of a subject's immune response relative to the effect obtained when the biologically active material is provided to the subject without the sphingoid-polyalkylamine conjugate(s).

Preferably, the amount would be sufficient to achieve effective immunization of a subject against a specific disease or disorder. It is noted that in accordance with some embodiments, the amount of the active material may be in the range from 1 μg to 125 μg, with a lipid to active material w/w ratio in the range of 10:1 to 1000:1 preferably in the range of 300:1 to 10:1 weight ratio.

The sphingoid-polyalkylamine conjugate(s) associated with the biologically active material may be administered in various ways. Non-limiting examples of administration routes include oral, subcutaneous (s.c.), intradermal (i.d.), intravenous (i.v.), intra-arterial (i.a.), transdermal (t.d.), intramuscular (i.m.), intraperitoneal (i.p.), intrarectal (i.r.), intra-vaginal, and intranasal (i.n.) administration, as well as by infusion techniques to the eye (intraocular).

Preferably modes of administration are the intranasal or intramuscular administrations. Preferred administration in accordance with the invention comprises intranasal or parenteral (e.g. intramuscular or subcutaneous routes).

Intranasal preparations may include excipients, which do not irritate the nasal mucosa, or do not inhibit severely the mucociliary function, and diluents such as water, brine. The intranasal preparations may include preservatives such as chlorobutanol and benzalkonium chloride, and also include surfactants for enhancing the absorption of biologically active material by nasal mucosa. The intranasal preparation may be adapted for administration by nasal spray, nasal drops, gel or powder etc. Aerosol formulations can be placed into pressurized acceptable propellants, such as propane, nitrogen, and the like. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or atomizer suitable carriers.

DESCRIPTION OF SOME NON LIMITING EXAMPLES

The following are experimental procedures and results providing non-limiting examples for the use of a conjugate in accordance with the invention.

Materials and Methods

Synthesis of N-palmitoyl D-erythro Sphingosyl Carbamoyl Spermine (CCS)

(i) N-palmitoylsphingosine (1.61 g, 3 mmol) was dissolved in dry tetrahydrofuran (THF) (100 ml) with heating. The clear solution was brought to room temperature and N,N′-disuccinimidyl carbonate (1.92 g, 7.5 mmol) was added. DMAP (4-dimethylamino pyridine) (0.81 g, 7.5 mmol) was added with stirring and the reaction further stirred for 16 hours. The solvent was removed under reduced pressure and the residue re-crystallized from n-heptane yielding 1.3 g (68%) of activated ceramide in the form of white powder m.p. 73-76° C.

(ii) Spermine (0.5 g, 2.5 mmol) and the activated ceramide (0.39 g, 0.5 mmol) were dissolved in dry dichloromethane with stirring and then treated with catalytic amount of DMAP. The solution was stirred at room temperature for 16 hours, the solvent evaporated and the residue treated with water, filtered and dried in vacuo, giving 0.4 μg (82%) of crude material which was further purified by column chromatography on Silica gel, using 60:20:20 Butanol:AcOH:H₂O eluent.

Synthetic CCS was isolated as its tri-acetate salt. It comprises a mixture of two isomers, the C-1 and C-3 isomers, as confirmed by ¹H-NMR and ¹³C-NMR spectrometry (see FIG. 9A in co-pending WO 2004/110980, incorporated herein by reference in its entirety).

The structure of the two isomers is illustrated in FIG. 1A (N-palmitoyl-D-erythro-sphingosyl-1-carbamoyl spermine, where the polyalkylamine (spermine) is attached via C-1, thus, at times referred to be the abbreviated C-1 CCS) and FIG. 1B (N-palmitoyl-D-erythro-sphingosyl-3-carbamoyl spermine, where the polyalkylamine (spermine) is attached via C-3, thus, at times referred to be N-palmitoyl D-erythro sphingosyl-3-carbamoyl spermine, the abbreviated C-3 CCS).

In the above and below description, unless otherwise stated, the product of the above synthetic procedure is used and hereinafter it referred to as the general term CCS.

(iii) for obtaining a quaternary ammonium salt within the compound, the product of step (ii) may be methylated with DMS or CH₃I.

Isolated Isomers N-palmitoyl D-erythro Sphingosyl Carbamoyl spermine (C-1 CCS or C-3 CCS)

It has been found that the above synthetic method provides a mixture of the two isomers, C-1 CCS and C-3 CCS. Isolated N-palmitoyl D-erythro sphingosyl-1-carbamoyl spermine and N-palmitoyl D-erythro sphingosyl-3-carbamoyl spermine were purchased from Biolab Ltd., Jerusalem, Israel. The use of an isolated isomer is specifically indicated, wherever appropriate.

At times, a mixture of the isolated isomers was also used and such a mixture is specifically referred to as the mixture of the isolated C-1 CCS and C-3 CCS isomers.

Influenza Antigens

A monovalent subunit antigen preparation derived from influenza A/New Caledonia/20/99-like (H1N1) strain was generously provided by Drs. Gluck and Zurbriggen, Berna Biotech, Bern, Switzerland. This preparation (designated herein HN) is comprised of 80-90% wt % hemagglutinin (H), 5-10 wt % neuraminidase (N) and trace amounts of NP and M1 proteins (this is referred to hereinbelow by the abbreviation HN or HA). A commercial trivalent subunit vaccine (Fluvirin®) for the 2003/2004 season containing HN derived from A/New Caledonia/20/99 (H1N1), A/Panama/2007/99 (H3N2) and B/Shangdong/7/97 was obtained from Evans Vaccines Ltd., Liverpool, UK. This vaccine was concentrated ˜×8 (Eppendorf Concentrator 5301, Eppendorf AG, Hamburg, Germany) prior to encapsulation.

In addition, for assessing immunogenicity of isolated isomers, the commercial trivalent vaccine Vaxigrip 2006-2007 (Aventis Pasteur) was used.

A whole inactivated virus was used in some experiments for in vitro stimulation.

Lipids

The phospholipids (PL), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), and dioleoyl phosphatidylethanolamine (DOPE) are from Lipoid GmbH, Ludwigshafen, Germany or from Avanti Polar Lipids (Alabaster, Ala., USA). In addition to DMPC (neutral) and DMPC/DMPG (9/1 mole ratio, anionic) liposomes, 6 formulations of cationic liposomes/lipid assemblies were prepared. The monocationic lipids dimethylaminoethane carbamoyl cholesterol (DC-Chol), 1,2-distearoyl-3-trimethylammonium-propane (chloride salt) (DSTAP), dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP), and dimyristoyl-3-trimethylammonium-propane (chloride salt) (DMTAP) are from Avanti Polar Lipids (Alabaster, Ala., USA). The monocationic lipid dimethyldioctadecylammonium bromide (DDAB) and cholesterol (Chol) are from Sigma. The polycationic sphingolipid N-palmitoyl-D-erythro-sphingosyl carbamoyl-spermine (acetate salt) (ceramide carbamoyl-spermine, CCS, i.e. the C-1 and/or C3 isomers) was produced as described above and the isolated isomers were purchased from Biolab Ltd., Jerusalem, Israel. Where indicated, the helper lipids (DOPE, Chol) were used at a lipid/helper lipid mole ratio of 1/1 to 4/1.

Animals

Specific pathogen-free (SPF) female BALB/c mice, 6-8 weeks old, and C57BL/6 mice, 18 month-old, were used (5-10 per group).

In addition, Sprague Dawley rats (females, 9 weeks old) were used (6 per group).

Ferrets (females, 6 months old) were used (8 per group).

Further, female Hartley strain guinea pigs, weighing about 300 grams each were used (from 5 to 7 per group)/

Animals were maintained under SPF conditions.

Preparation of Lipid Assemblies

For the formation of lipids assemblies, lipids (10-30 mg) were dissolved in 1 ml tertiary butanol.

For CCS preparation, three procedures were used to form CCS and/or cholesterol solutions:

-   -   1. an amount of CCS was dissolved with DDW and the helper lipid,         cholesterol, was dissolved in tertiary butanol;     -   2. each of the lipids (CCS and the helper lipid) were dissolved         (separately) in tertiary butanol; or     -   3. each of the lipids were dissolved in Dichloromethane:Methanol         (2:1).

The CCS solution and the cholesterol solution were mixed to obtain a mixture with CCS/Cholesterol mole:mole ratio 3:2.

For all lipids, the lipid solutions were sterilized by filtration (GF92, Glasforser, Vorfilter no. 421051, Schleicher & Schuell, Dassel, Germany). The sterile lipid solution was frozen at −70° C. (for the CCS dissolved with Dichloromethane:Methanol, the solution was evaporated with a gentle stream of nitrogen) then lyophilized for 24 h to complete dryness. The dried lipids could be stored at 4° C. for >2 years without significant (<10%) lipid degradation or loss of “encapsulation” capability. Upon need, the lipid powder was hydrated with the antigen solution (in PBS pH 7.2) at a lipid:antigen (protein) w/w ratio of 3/1 to 800/1. The antigen solution was added stepwise in small increments and vortexed vigorously after each addition, up to a final volume of 0.5-1 ml. In some experiments, the dried lipids were hydrated with PBS and the preformed “empty” lipid assemblies were mixed with the antigen solution. The mixture was vortexed for 1-2 min and used as is within 30-60 min.

Encapsulation of Influenza Antigens in Liposomes/Lipid Assemblies

HN antigens (see above) were encapsulated in large (mean diameter 0.1-5 μm) heterogeneous (unsized) vesicles.

To determine “encapsulation” efficiency, two procedures were used, depending on the formulation, resulting in ≧80% separation between the free antigen and the lipid-associated antigen. For all vaccine formulations, except CCS, the following separation technique was used. The lipid assemblies (1-30 mg lipid) containing the HN antigen (50-100 μg protein) were suspended in 0.5 ml PBS and carefully loaded over 0.5 ml of D₂O (99.9%, Aldrich Chemical Co., Milwaukee, Wis., USA). The sample was then centrifuged for 1 h at 30° C. at 45,000 rpm. The free, non-encapsulated HN precipitates while the assembled (liposomal) HN and protein-free lipid assemblies/liposomes remain in the supernatant. The entire supernatant was collected and the assemblies/liposomes were dissolved by adding 0.2 ml of warm 10% Triton X-100 to both the supernatant and the pellet fractions. Protein concentration in both fractions was determined by the modified Lowry technique. For the CCS formulations, the CCS was suspended in 0.5 ml of Ag solution and then carefully loaded over 0.5 ml D₂O. The sample was then centrifuged for 10 min at 20° C. at 10,000 rpm. The large liposomes were present in the middle phase while the little ones may precipitate. The free HN remains in the upper phase. The 3 fractions were collected separately. Lipid dissolution was done using boiled 20% Triton X-100 to give a final concentration of 10% Triton and protein determination in the 3 fractions was carried out by the modified Lowry technique. In both separation techniques, the overall recovery of the HN antigens was >95%.

In order to test the immunogenicity of isolated CCS isomers or of the mixture of the isolated isomers, a preparation of the isolated CCS (or mixture thereof) and cholesterol (CCS/Cholesterol mole:mole ratio 3:2) was prepared in two methods: DDW/T-Butanol and Dichloromethane/methanol (described above).

Immunization

Immunization of Mice:

Free (F-HN) and assembled/liposomal (Lip-HN) vaccines, 0.25-4 μg antigen/strain/dose and 0.075-1.2 mg lipid/dose, were administered either once intramuscularly (i.m., in 30 μl), once or twice intranasally (i.n., in 5-50 μl per nostril) spaced 3, 7 or 14 days apart, or twice orally (in 50 μl) spaced 1 week apart. When assessing the immunogenicity of the isolated isomers or the mixture of isolated isomers (mole ratio of C-1 CCS to C-3 CCS being 80:20), BALB/C mice (female 8 weeks) were vaccinated once i.m. with 6 μg total HA (Vaxigrip) with or without the isomer, the isomers being mixed with cholesterol as described above. In the latter experiment, the lipid/antigen ratio was 150:1; serum antibody titer against A/New Calendonia virus was conducted 4 weeks following vaccination using the HI assay as described below.

In all cases of intranasal (i.n.) administration mice were lightly anesthetized with 0.15 ml of 4% chloral hydrate in PBS given intraperitoneally. For oral vaccination mice were treated orally with 0.5 ml of an antacid solution (8 parts Hanks' balanced salt solution+2 parts 7.5% sodium bicarbonate) 30 min prior to vaccination. Cholera toxin (CT, Sigma, USA), 1 μg/dose, was used in some experiments as a standard mucosal adjuvant for comparison. In two experiments, CpG-ODN (ODN 1018, generously provided by Dr. E. Raz, University of California, San Diego, Calif., USA), free and liposomal, 10 μg/dose, was used as an adjuvant.

Immunization of Rats:

Sprague Dawley rats (females, 9 weeks old) were vaccinated intramuscularly (i.m.) once with 12 μg total HA (Vaxigrip) with/without CCS/C (3:2) [the CCS comprising the mixture of isomers synthesized as described above]. Lipid:antigen ratio 150:1. Serum antibody titer against A/New Caledonia virus was followed for 8 weeks by hemagglutination inhibition (HI) assay.

Immunization of Ferrets:

Ferrets (females, 6 months old) were vaccinated i.m. twice on day 0 and day 14 with CCS/C alone (negative control), with free antigen (F-HA) or with CCS/C-HA 15 μg per strain (total 45 μg, Vaxigrip). CCS/C-HA vaccine was also tested at a dose of 5 μg per strain.

Immunization of Guinea Pigs:

Guinea pigs were vaccinated s.c. once on day 0 with free PA antigen (F-PA) or with CCS/C-PA at an amount of 25 μg PA antigen (in 100 μl) or with STI (positive control, 0.5 ml).

Assessment of Humoral Responses

Sera, lung homogenates and nasal washes were tested, individually or pooled, 4-6 weeks post-vaccination, starting at 1/10 or 1/20 sample dilution. Hemagglutination inhibiting antibodies were determined by the standard hemagglutination inhibition (HI) assay. Mice with HI titer ≧40 (considered a protective titer in humans) were defined as seroconverted. Antigen-specific IgG1, IgG2a, IgA and IgE levels were measured by ELISA. The highest sample dilution yielding absorbance of 0.2 OD above the control (antigen+normal mouse serum, OD <0.1) was considered the ELISA antibody titer [for ELISA protocol, see materials and methods in Babai, S. Samira, Y. Barenholz, Z. Zakay-Rones and E. Kedar, Vaccine 17 (1999) (9/10), pp. 1239-1250].

In some experiments (see below), the humoral response was tested at other time points following vaccination.

In the description above and below, whenever referring to ELISA and unless otherwise stated, the ELISA was performed as described by Babai et al., mutatis mutandis.

Assessment of Cellular Responses

Splenocytes obtained at 5-6 weeks after vaccination were tested for proliferative response, IFNγ and IL-4 production, and cytotoxic activity, following in vitro stimulation with the antigen. Cultures were carried out at 37° C. in enriched RPMI 1640 or DMEM medium supplemented with 5% (for proliferation, cytokines) or 10% (for cytotoxicity) fetal calf serum (FCS), with (for cytotoxicity) or without 5×10⁻⁵M 2-mercaptoethanol. Cell cultures were performed as follows: (i) Proliferation: 0.5×10⁶ cells per well were incubated in U-shaped 96-well plates, in triplicate, with or without the antigen (0.5-5 μg per well), in a final volume of 0.2 ml. After 72-96 h, cultures were pulsed with 1 μCi ³H-thymidine for 16 h. Results are expressed in Δcpm=(mean counts per minute of cells cultured with antigen)−(mean counts per minute of cells cultured without antigen). (ii) Cytokines: 2.5×10⁶ to 5×10⁶ cells per well were incubated in 24-well plates, in duplicate, with or without the antigen (5-10 μg per well), in a final volume of 1 ml. Supernatants were collected after 48-72 h and tested by ELISA for murine IFNγ and IL-4 using the Opt EIA Set (Pharmingen, USA). (iii) Cytotoxicity: Responding splenocytes (2.5×10⁶) were incubated as in (ii) for 7 days together with an equal number of stimulating BALB/C splenocytes that had been infected with the X/127(H1N1) influenza virus (see below). For infection, the splenocytes were incubated, with occasional stirring, for 3 h at 37° C. in RPMI 1640 medium (without FCS) with 150 hemagglutination units/1×10⁶ splenocytes of the virus, followed by washing. Subsequently, the primed effector cells were restimulated for 5 days with infected, irradiated (3,000 rad) splenocytes at an effector/stimulator cell ratio of 1/4 in the presence of 10 IU/ml of rhIL-2. Cytotoxicity was measured using the standard 4 h ⁵¹Cr release assay at an effector/target cell ratio of 100/1. The labeled target cells used were unmodified P815 and P815 pulsed for 90 min at 37° C. with the HA2 189-199 peptide (IYSTVASSLVL, 20 μg/1×10⁶ cells).

Determination of Protective Immunity

Mice were anesthetized and 25 μl of live virus suspension per nostril, ˜10⁷ EID 50 (egg-infectious dose 50%), was administered, using the reassortant virus X-127 (A/Beijing/262/95 (H1N1)×X-31 (A/Hong Kong/1/68×A/PR/8/34), which is infectious to mice and cross-reactive with A/New Caledonia. The lungs were removed on day 4, washed thrice in cold PBS, and homogenized in PBS (1.5 ml per lungs per mouse, referred to as 1/10 dilution). Homogenates of each group were pooled and centrifuged at 2000 rpm for 30 min at 4° C. and the supernatants collected. Serial 10-fold dilutions were performed and 0.2 ml of each dilution was injected, in duplicate, into the allantoic sac of 11-day-old embryonated chicken eggs. After 48 h at 37° C. and 16 h at 4° C., 0.1 ml of allantoic fluid was removed and checked for viral presence by hemagglutination (30 min at room temperature) with chicken erythrocytes (0.5 wt. %, 0.1 ml). The lung virus titer is determined as the highest dilution of lung homogenate producing virus in the allantoic fluid (positive hemagglutination).

The ferrets were challenged intranasally with the homologous Influenza A/New Caledonia/20/99 virus [H1N1] (4.93 TCID₅₀/ml diluted 1/100 v/v) on day 28 post-immunization. Following challenge, nasal washes were performed daily for 9 days post-infection, for analysis of virus shedding from the nasal mucosa. Viral shedding from nasal washes was determined by titration on MDCK cells.

MDCK cells were plated and grown for 1-2 days until they were observed to be between 50% and 70% confluent. Samples were added to the cells in duplicates, and diluted at a 1 in 10 (v/v) serial dilution, to give a dilution range of 10-1 to 10-6 (v/v). A known titre of virus was used as a control in the assay. The cells were incubated at 37° C. with 5% CO2. Once the control titration was observed to have cytopathic effect (CPE) in the expected virus control wells (approximately 3-4 days), the hemagglutination assay was performed on a sample of the supernatants.

Biodistribution and Pharmacokinetics of Various Fluorescently-Labeled Lipid Formulations and Radioactively-Labeled HN Antigen

Mice were vaccinated once intranasally (i.n.) with lissamine-rhodamine labeled liposomes, either empty or associated with trivalent subunit influenza vaccine (HN) in a volume of 20 μl. After 1, 5 or 24 hours, mice were sacrificed and various organs were removed. The organs were stored at −20 deg overnight, and the next morning homogenized in lysis buffer. 0.2 ml the subsequent homogenate was transferred to Eppendorf tubes, 0.8 mL of isopropanol was added, and spun for 15 minutes to release fluorescent probe into the supernatant. 50 uL of the supernatant was loaded onto a 384 black plate and the fluorescence was read (Em: 545, Ex: 596).

In a further assay, 450 μg of trivalent HN vaccine (in 5 mL) were dialysed against DDW (to remove salt) and then concentrated ×1000 to 5 μL. The protein was then diluted in 0.1M borate buffer (pH 8.5) to a stock solution of 450 μg in 15 μL. The protein was then labeled with ¹²⁵I using the Bolton Hunter reagent, according to the manufacturer's instructions. Mice were immunized i.n. with the ¹²⁵I-labeled HN (2 μg), either free or liposome encapsulated, and at 1, 5 and 24 hrs the mice were sacrificed, and various organs (see FIG. 3) were removed into vials and read in a γ-counter calibrated for ¹²⁵I.

Results

Characterization of HN Antigen-Loaded Cationic Liposomes

Efficiency of encapsulation of HN (a commercial preparation of hemagglutinin and neuraminidase derived from influenza viruses) loaded onto various cationic liposomal formulations, at different lipid/protein w/w ratios (3/1-300/1), and with or without cholesterol (Chol) was tested. Table 1 shows the results of such an experiment, using the cationic lipids DOTAP and CCS. TABLE 1 The effect of the lipid (DOTAP, CCS)/protein ratio and cholesterol (Chol) on HN encapsulation efficiency DOTAP/HN DOTAP/Chol % HN CCS/HN CCS/Chol % HN w/w ratio mole ratio encapsulation w/w ratio mole ratio encapsulation 300/1 1/1 93 300/1 1/0 73 100/1 1/1 90 100/1 1/0 64  50/1 1/1 90  30/1 1/0 38  30/1 1/1 88  10/1 1/0 1  10/1 1/1 79  3/1 1/1 35 100/1 1/0 90 300/1 3/2 71 100/1 1/1 92 100/1 3/2 64 100/1 2/1 89  30/1 3/2 41 100/1 4/1 80  10/1 3/2 0 A monovalent vaccine was used for DOTAP and a trivalent vaccine (concentrated) for CCS.

The percentage of antigen loading for DOTAP was 80-90% using a lipid/protein w/w ratio of 30/1 to 300/1, with and without Chol, decreasing to 79% and 35% at 10/1 and 3/1 w/w ratios, respectively. The addition of Chol to the formulation did not affect loading at DOTAP/Chol mole ratios of 1/1 and 2/1, with slightly lower encapsulation (80%) at a ratio of 4/1. For CCS, with or without Chol, the loading efficiency was lower (64-73% at w/w ratio of 100/1-300/1).

HN association with the liposomes upon simple mixing of the soluble antigen with preformed empty liposomes was also determined. In such cases, 40-60% of the antigen was associated with the liposomes using a lipid/protein w/w ratio of 100/ to 300/1, regardless of the formulation.

These finding, collectively, indicate very high loading efficiency (>60%) using a simple and fast (5 min.) procedure in all formulations. Furthermore, even preformed liposomes in aqueous suspension were capable of effectively associating with the influenza virus surface antigens.

The immunogenicity of the various lipid/antigen w/w ratios (with or without the addition of cholesterol) was also evaluated.

In a first experiment sera levels of HI, IgG1 and IgG2a antibodies following i.n. vaccination of young (2-month-old) BALB/c mice with HN-loaded neutral, anionic or cationic liposomes were determined (Table 2A). The HN antigen was a monovalent subunit vaccine derived from the A/New Caledonia (H1N1) strain. In the same experiment, lung and nasal levels of HI, IgG1, IgG2a and IgA antibodies, and INFγ levels produced by spleen cells, were also tested (Tables 2B and 2C). TABLE 2A Sera levels of HI, IgG1 and IgG2a antibodies (i.n. administration) Group Serum (n = 5) Vaccine HI (mean ± SD) IgG1 IgG2a 1 PBS 0 0 0 2 F-HN 0 55 0 3 Lip (DMPC)-HN   3 ± 7 (0) 150 0 (Neutral) 4 Lip (DMPC/DMPG)-   6 ± 13 (20) 500 0 HN (Anionic) 5 Lip (DC-Chol:DOPE)-  18 ± 7 (0) 0 0 HN 6 Lip (DSTAP:Chol)-  28 ± 29 (40) 20 0 HN 7 Lip (DDAB:Chol)-HN  136 ± 32 (100) 100 0 8 Lip (DOTAP:Chol)-  576 ± 128 (100) 15000 730 HN 9 Lip (DMTAP:Chol)-  672 ± 212 (100) 30000 470 HN 10 Lip (CCS:Chol)-HN 2368 ± 1805 (100) 30000 9000 11 F-HN + CT (1 μg) 1664 ± 572 (100) 55000 7000 F-HN, free antigen; Chol, cholesterol; CT, cholera toxin. Groups 5-10 are cationic liposomes. In parentheses, % seroconversion − % of mice with HI titer ≧40.

In particular, a comparison was made between neutral (DMPC), anionic (DMPC/DMPG, 9/1 mole ratio) and cationic (6 formulations) assemblies (Lip) encapsulating the HN antigens to induce local and systemic responses following two i.n. administrations. For all formulations, the lipid/HN w/w ratio was 300/1, and the cationic lipid/Chol or cationic lipid/DOPE mole ratio was 1/1. Free antigen (F-HN) and F-HN co-administered with cholera toxin (CT, 1 μg) as an adjuvant were tested in parallel. The vaccine was given on days 0 and 7, 3 μg/dose (10 μl per nostril), and the responses were determined 4 (HI) and 6 (Elisa) weeks after the second vaccine dose. TABLE 2B lung and nasal wash levels of IgG1, IgG2a and IgA antibodies Group Lung Nasal (n = 5) Vaccine IgG1 IgG2a IgA IgG1 IgG2a IgA 1 PBS 0 0 0 0 0 0 2 F-HN 0 0 0 0 0 0 3 Lip (DMPC)-HN (Neutral) 30 0 0 0 0 0 4 Lip (DMPC/DMPG)-HN (Anionic) 40 0 0 0 0 0 5 Lip (DC-Chol:DOPE)-HN 0 0 0 0 0 0 6 Lip (DSTAP:Chol)-HN 0 20 0 0 80 0 7 Lip (DDAB:Chol)-HN 0 80 30 0 30 0 8 Lip (DOTAP:Chol)-HN 730 1050 170 40 180 30 9 Lip (DMTAP:Chol)-HN 470 3000 30 15 80 70 10 Lip (CCS:Chol)-HN 9000 30000 1900 15000 30 300 11 F-HN + CT (1 μg) 7000 10000 1800 40 120 30

TABLE 2C Spleen INFγ levels (pg/ml) Group Spleen (n = 5) Vaccine IFNγ (pg/ml) 1 PBS 1800 2 F-HN 1400 3 Lip (DMPC)-HN (Neutral) 4200 4 Lip (DMPC/DMPG)-HN (Anionic) 4000 5 Lip (DC-Chol:DOPE)-HN 4900 6 Lip (DSTAP:Chol)-HN 2300 7 Lip (DDAB:Chol)-HN 3100 8 Lip (DOTAP:Chol)-HN 8000 9 Lip (DMTAP:Chol)-HN 7800 10 Lip (CCS:Chol)-HN 10200 11 F-HN + CT (1 μg) 5200

As shown in Tables 2A-2C, the free antigen, as well as the neutral and anionic Lip-HN were virtually ineffective intranasal vaccines. In contrast, the cationic Lip-HN, particularly those designated DOTAP-HN, DMTAP-HN and CCS-HN, evoked a robust systemic and mucosal humoral response, with high levels of IgG1, IgG2a and IgA antibodies, namely a mixed Th1+Th2 response. No IgE antibodies were defected. The cationic liposomal vaccines comprising DOTAP-HN, DMTAP-HN and CCS-HN also induced high levels of IFNγ (but not IL-4) in antigen-stimulated spleen cells. The responses produced by CCS-HN were even stronger than those induced by F-HN adjuvanted with CT. Based on these findings, only the cationic liposomal formulations: DOTAP-HN, DMTAP-HN and CCS-HN were further used.

In a second experiment, the effect of lipid/HN w/w ratio on the immunogenicity of HN-loaded cationic liposomes and of preformed liposomes, simply mixed with the soluble antigen, was determined. The data shown in Tables 3A-3C indicate that all three formulations induced a strong systemic (serum) and local (lung) response, and that lowering the lipid HN w/w ratio below 100/1 markedly reduced the antibody response. TABLE 3A Serum levels of HI, IgG1, IgG2a and IgA antibodies Lipid/HN Serum No. Vaccine (n = 5) w/w ratio HI IgG1 IgG2a IgA 1 F-HN 0 0 0 0 2 Lip (DOTAP)-HN 300/1 496 ± 495 (100) 15000 450 0 3 100/1 196 ± 119 (100) 5000 280 0 4  30/1  36 ± 50 (80) 1000 200 0 5  10/1  28 ± 18 (60) 600 30 0 6  3/1 0 20 0 0 7 Lip (DMTAP)-HN 300/1 388 ± 260 (100) 2500 250 0 8 100/1 208 ± 107 (100) 2200 600 0 9  50/1 130 ± 118 (80) 850 150 0 10  30/1  48 ± 71 (40) 450 0 0 11  10/1  24 ± 35 (40) 120 0 0 12 Lip (CCS)-HN 300/1 560 ± 480 (100) 2000 1800 200 13 100/1 752 ± 504 (100) 6500 6000 0 14  50/1 272 ± 156 (100) 1900 700 0 15  30/1 112 ± 125 (80) 650 400 0 16  10/1  52 ± 68 (40) 275 440 0 17 F-HN + CT (1 μg) — 896 ± 350 (100) 30000 8000 120 18 F-HN + Lip (DOTAP) 300/1 864 ± 446 (100) 5000 1500 0 19 F-HN + Lip (DMTAP) 300/1 320 ± 226 (100) 1900 400 0 20 F-HN + Lip (CCS) 300/1 704 ± 525 (100) 30000 5000 500 In groups 18-20 preformed liposomes were mixed with the soluble antigen.

TABLE 3B Lung levels of HI, IgG1, IgG2a and IgA Lipid/ HN w/w Lung No. Vaccine (n = 5) ratio HI IgG1 IgG2a IgA 1 F-HN 0 0 0 0 2 Lip (DOTAP)-HN 300/1 40 600 85 30 3 100/1 40 500 20 0 4  30/1 30 250 35 0 5  10/1 20 250 0 0 6  3/1 10 20 0 0 7 Lip (DMTAP)-HN 300/1 0 5500 200 1200 8 100/1 0 7000 350 0 9  50/1 0 4500 250 0 10  30/1 0 1500 110 0 11  10/1 0 500 0 0 12 Lip (CCS)-HN 300/1 80 12500 3000 20000 13 100/1 80 7000 5500 65000 14  50/1 40 5500 900 20000 15  30/1 0 1500 200 0 16  10/1 0 500 200 0 17 F-HN + CT (1 μg) — 80 45000 2250 3000 18 F-HN + Lip 300/1 0 6000 500 1200 (DOTAP) 19 F-HN + Lip 300/1 0 3750 225 1500 (DMTAP) 20 F-HN + Lip (CCS) 300/1 80 35000 3000 80000

TABLE 3C Spleen INFγ levels (pg/ml) Lipid/HN Spleen No. Vaccine (n = 5) w/w ratio IFNγ (pg/ml) 1 F-HN 4500 2 Lip (DOTAP)-HN 300/1 9780 3 100/1 42220 4  30/1 20440 5  10/1 20400 6  3/1 27780 7 Lip (DMTAP)-HN 300/1 3500 8 100/1 5850 9  50/1 3400 10  30/1 3050 11  10/1 Not done 12 Lip (CCS)-HN 300/1 8000 13 100/1 8250 14  50/1 10650 15  30/1 3500 16  10/1 Not done 17 F-HN + CT (1 μg) — 22800 18 F-HN + Lip (DOTAP) 300/1 3400 19 F-HN + Lip (DMTAP) 300/1 5700 20 F-HN + Lip (CCS) 300/1 4100

The superiority of Lip CCS-HN vaccine over the other vaccine formulations is again seen as reflected by the high levels of serum and lung IgG2a and IgA antibodies (groups 12-16). Interestingly, simple mixing of soluble antigen with preformed liposomes generated very potent vaccines (groups 18-20) that are equal to liposomes encapsulating the antigen. This suggests that real encapsulation of the antigen may not be necessary for the adjuvanticity of the cationic assemblies/liposomes.

In a further experiment the effect of cholesterol on the immunogenicity of the HN-loaded liposomes was tested. Tables 4A-4C show the results of this experiment, indicating that the addition of Cholesterol slightly reduced the systemic HI response to DOTAP-HN at 2/1 and 4/1 mole ratios (groups 4, 5), but not at a 1/1 mole ratio (group 3), and moderately enhances the overall response to DMTAP-HN at all ratios (groups 7-9) and the local (lung) response CCS-HN at a 1/1 ratio (group 11). TABLE 4A Serum levels of HI, IgG1, IgG2a and IgA antibodies Vaccine Cat lipid/Chol Serum No. (n = 5) mole ratio HI IgG1 IgG2a IgA 1 F-HN — 0 0 0 0 2 Lip (DOTAP)-HN 1/0  320 ± 0 (100) 15000 450 0 3 Lip (DOTAP:Chol)- 1/1  496 ± 295 (100) 15000 450 0 HN 4 2/1  168 ± 216 (100) 7000 800 0 5 4/1  195 ± 111 (100) 15000 250 0 6 Lip (DMTAP)-HN 1/0  320 ± 188 (100) 20000 290 0 7 Lip (DMTAP:Chol)- 1/1  672 ± 419 (100) 30000 300 0 HN 8 2/1  576 ± 368 (100) 25000 650 0 9 4/1  608 ± 382 (100) 30000 600 0 10 Lip (CCS)-HN 1/0 2560 ± 1568 (100) 30000 7000 100 11 Lip (CCS:Chol)-HN 1/1 2368 ± 1805 (100) 30000 9000 100 12 F-HN + CT (1 μg) — 1664 ± 572 (100) 55000 7000 20

TABLE 4B Lung levels of HI, IgG1, IgG2a and IgA antibodies Cat lipid/ Chol Vaccine mole Lung No. (n = 5) ratio HI IgG1 IgG2a IgA 1 F-HN — 0 0 0 0 2 Lip (DOTAP)-HN 1/0 40 900 85 25 3 Lip (DOTAP:Chol)- 1/1 40 600 80 30 HN 4 2/1 40 680 180 22 5 4/1 60 720 50 60 6 Lip (DMTAP)-HN 1/0 60 1000 40 0 7 Lip (DMTAP:Chol)- 1/1 120 3000 30 15 HN 8 2/1 160 2500 160 200 9 4/1 80 4000 100 150 10 Lip (CCS)-HN 1/0 640 30000 1500 9000 11 Lip (CCS:Chol)-HN 3/2 1280 30000 1900 15000 12 F-HN + CT (1 μg) — 20 10000 1800 1000

TABLE 4C Spleen INFγ levels (pg/ml) Vaccine Cat lipid/Chol Spleen No. (n = 5) mole ratio IFNγ (pg/ml) 1 F-HN — 7430 2 Lip (DOTAP)-HN 1/0 7480 3 Lip (DOTAP:Chol)-HN 1/1 9780 4 2/1 12870 5 4/1 9330 6 Lip (DMTAP)-HN 1/0 8520 7 Lip (DMTAP:Chol)-HN 1/1 10900 8 2/1 8560 9 4/1 7490 10 Lip (CCS)-HN 1/0 15550 11 Lip (CCS:Chol)-HN 3/2 13780 12 F-HN + CT (1 μg) — 11110

The immunogenicity of CCS-HN vaccine was also evaluated in aged (18 month) C57BL/6 mice following intramuscular (once on day 0) or intranasal (twice, days 0 and 7) administration of 1 μg and 2 kg, respectively, of subunit (HN) vaccine (derived from A/Panama [H3N2] virus). The lipid assemblies were composed of CCS/cholesterol (3:2 molar ratio) and the lipid/HN w/w ratio was 200:1. As opposed to zero activity of the commercial vaccine, the CCS-HN vaccine evoked high levels of serum HI and IgG2a antibodies (tested at 4 weeks post vaccination) and lung (tested at 6 weeks post vaccination) IgG2a and IgA antibodies, as can be seen in Tables 5A and 5B (the data show mean titers). TABLE 5A Serum levels of HI, IgG1, IgG2a and IgA in aged mice Vaccine^(a) Serum No. (n = 5) HI IgG1 IgG2a 1 PBS i.n. × 2 0 0 0 2 F-HN i.m. × 1 0 15 0 3 F-HN i.n. × 2 0 0 0 4 Lip (CCS)-HN i.n. × 2 80 130 350

TABLE 5B Lung levels of IgG1, IgG2a and IgA in aged mice Vaccine Lung No. (n = 5) IgG1 IgG2a IgA 1 PBS i.n. × 2 0 0 0 2 F-HN i.m. × 1 0 0 0 3 F-HN i.n. × 2 0 0 0 4 CCS-HN i.n. × 2 0 180 840

In addition, the induction of cellular responses by the various vaccine formulations was tested. In particular, young mice were immunized i.n. (days 0, 7) with various cationic liposomal formulations and the splenocyte cellular responses—cytotoxicity, proliferation and IFNγ production—were measured 6 weeks after vaccination. In the experiment, the results of which are shown in Table 6, a comparison was made between HN-loaded liposomes (groups 3-10) and free antigen (F-HN) given alone (group 2) or admixed with preformed empty liposomes (groups 11-13). The immunogenicity of Lip (DMTAP)-HN and Lip (CCS)-HN prepared at varying lipid/HN w/w ratios (30/1-300/1) was also determined. TABLE 6 Induction of cellular responses by cationic liposomes administered i.n. Lipid/HN % cytotoxicity Proliferation IFNγ No. Vaccine w/w ratio P815 + peptide P815 Δcpm (mean) (pg/ml) 1 PBS — 6 4 7010 1900 2 F-HN — 8 5 7700 4500 3 Lip (DMTAP)-HN 300/1 16 13 10960 3500 4 100/1 9 9 12870 5850 5  50/1 3 2 17670 3400 6  30/1 3 2 17920 3050 7 Lip (CCS)-HN 300/1 4 2 20370 8000 8 100/1 21 7 24870 8250 9  50/1 6 3 20980 10650 10  30/1 8 5 11510 3500 11 F-HN + Lip (DOTAP) 300/1 17 4 19390 3400 12 F-HN + Lip (DMTAP) 300/1 17 7 11850 5700 13 F-HN + Lip (CCS) 300/1 16 8 19270 4100

Preferential cytotoxicity against the specific target cells (P815 pulsed with the influenza peptide) was obtained only with CCS-HN at a lipid/HN w/w ratio of 100/1 (group 8) and with all the three preformed liposomes (DOTAP, DMTAP and CCS) co-administered with free antigen. The maximum proliferative response was observed with DMTAP-HN at lipid/HN w/w ratios of 50/1 and 30/1 and with CCS-HN at 300/1, 100/1 and 50/1 ratios. The proliferative and cytotoxic responses elicited by the most efficacious liposomal formulations were 2-3 times greater than those induced by free antigen.

These findings suggest that as compared with the humoral response (Table 3), where the highest levels of all types of antibodies measured were obtained at lipid/HN w/w ratios of 100/1-300/1, lower w/w ratios (e.g. 30/1-100/1) may be optimal for the cellular responses. Moreover, whereas DMTAP-HN elicits a strong humoral response, this formulation is a poor inducer of cytotoxic activity, as compared with CCS-HN. Interestingly, vaccination with mixtures of free antigen with preformed cationic liposomes (all three formulations) in suspension evokes good cellular responses that are similar in magnitude to those induced by the encapsulated antigen. Thus, simple mixing of free antigen with preformed cationic liposomes may be sufficient to induce both strong humoral (Table 3A-3C) and cellular (Table 6) responses.

In yet a further experiment, the results of which are shown in Tables 7A-7C, a comparison was made between 1 i.m. dose, 1 or 2 i.n. doses and 2 oral doses of a monovalent HN-loaded cationic liposomes comprising DOTAP, DMTAP or CCS with regard to immunogenicity and induction of protective immunity to live virus challenge. In this experiment, the lipid/HN w/w ratio was 300/1 and the cationic lipid/Chol ratio was 1/1 for DOTAP and DMTAP systems and 3/2 for CCS system. Of the three routes, i.n. administration twice generates the strongest humoral (systemic and mucosal) and cellular response and protective immunity. Of the 3 formulations, CCS induces the highest response, particularly with regard to IgG2a and IgA antibodies. TABLE 7A Serum levels of HI, IgG1, IgG2a and IgA Vaccine Serum No. (n = 10) Route HI IgG1 IgG2a IgA 1 PBS 0 0 0 0 2 F-HN i.m. × 1  60 ± 37 (70) 1000 40 0 3 oral × 2 0 0 0 0 4 i.n. × 1 0 0 0 0 5 i.n. × 2 0 55 0 0 6 Lip (DOTAP/Chol)-HN i.m. × 1  424 ± 141 (100) 21000 5500 0 7 oral × 2 0 0 0 0 8 i.n. × 1  40 ± 28 (50) 450 80 0 9 i.n. × 2  409 ± 172 (100) 25000 1300 60 10 Lip (DMTAP/Chol)-HN i.m. × 1  768 ± 211 (100) 24000 8000 0 11 oral × 2 0 0 0 0 12 i.n. × 1  10 ± 10 (0) 300 60 0 13 i.n. × 2  532 ± 763(100) 10500 380 50 14 Lip (CCS/Chol)-HN i.m. × 1  864 ± 1100 (100) 25000 10000 0 15 oral × 2 0 0 16 i.n. × 1  34 ± 50 (20) 1000 30 0 17 i.n. × 2 2289 ± 1576 25000 20000 400 (100) 18 F-HN + CT (1 μg) i.n. × 2  756 ± 650 (100) 21000 15000 20

TABLE 7B Lung antibodies Vaccine Lung No. (n = 5) Route HI IgG1 IgG2a IgA 1 PBS 0 0 0 0 2 F-HN i.m. × 1 0 80 0 0 3 oral × 2 0 0 0 0 4 i.n. × 1 0 0 0 0 5 i.n. × 2 0 70 20 0 6 Lip (DOTAP/Chol)- i.m. × 1 40 900 500 0 HN 7 oral × 2 0 0 0 0 8 i.n. × 1 0 50 20 0 9 i.n. × 2 120 10000 1000 350 10 Lip (DMTAP/Chol)- i.m. × 1 20 900 150 0 HN 11 oral × 2 0 0 0 0 12 i.n. × 1 0 35 20 0 13 i.n. × 2 240 20000 700 2200 14 Lip (CCS/Chol)-HN i.m. × 1 60 3500 900 0 15 oral × 2 0 0 0 0 16 i.n. × 1 0 120 0 35 17 i.n. × 2 360 30000 5000 20000 18 F-HN + CT (1 μg) i.n. × 2 240 22000 2500 1800

TABLE 7C Cellular response and protective immunity Spleen Lung Vaccine Δcpm IFNγ Virus titer No. (n = 5) Route (mean) (pg/ml) (log 10) 1 PBS 1641 0 7 2 F-HN i.m. × 1 1909 0 4 3 oral × 2 2253 0 ND 4 i.n. × 1 669 0 ND 5 i.n. × 2 2813 0 5 6 Lip (DOTAP/Chol)-HN i.m. × 1 3452 3300 0 7 oral × 2 0 1150 ND 8 i.n. × 1 482 1900 ND 9 i.n. × 2 8391 3200 0 10 Lip (DMTAP/Chol)-HN i.m. × 1 5632 0 1 11 oral × 2 553 0 ND 12 i.n. × 1 1277 0 ND 13 i.n. × 2 7331 3150 0 14 Lip (CCS/Chol)-HN i.m. × 1 6196 5750 0 15 oral × 2 476 550 ND 16 i.n. × 1 1705 6250 ND 17 i.n. × 2 4912 15500 0 18 F-HN + CT (1 μg) i.n. × 2 1933 5650 0

In the experiment described in Tables 8-10, a commercial trivalent vaccine was tested and a comparison was made between a single intranasal (i.n.) CCS-based vaccine dose (using 2 or 4 μg of antigen [HN] of each viral strain) and two i.n. vaccine doses (2 μg/strain/dose), given at 3, 7 or 14 day intervals between administrations. The lipid assemblies were composed of CCS/Chol (cholesterol) at a 3/2 mole ratio, and the lipid/HN w/w ratio was 100/1 for all formulations. As controls, the standard trivalent commercial vaccine (HN) was administered either alone or combined with 1 μg cholera toxin (CT), used as a mucosal adjuvant. Sera, lung homogenates and nasal washes were tested 5-6 weeks after the first vaccine dose for HI antibodies (Table 8), as well as for antigen-specific IgG1, IgG2a, IgA and IgE antibodies (Table 9). In addition, 5 mice from selected groups were challenged i.n. with live virus (using the mouse adapted reassortant X-127 virus) and protection was assessed by quantifying lung virus titer 4 days later (Table 10).

As opposed to the poor or no immunogenicity of the commercial flu vaccine (HN) (groups 2-6), CCS/Chol-flu vaccine induced high titers of all types of antibodies tested (except for IgE which was undetected), especially against the two A virus strains (groups 8-11; Tables 8, 9). For the 2-dose regimen, a 1-week interval appears to be the optimal (gr. 10). For the single dose regimen, 4 μg antigen, but not 2 μg (gr. 8 vs. gr. 7), induced high titers of serum HI, IgG1 and IgG2a antibodies and lung IgG1 antibodies. However, in comparison with the 2-dose regimen, the 1-dose regimen did not elicit lung IgG2a and IgA antibodies or nasal antibodies (Table 9).

In the protection assay (Table 10), the CCS-flu vaccine administered i.n. either once (4 μg) or twice (2 μg/dose) afforded full protection against viral infection (6 log reduction in lung virus titer) whereas the standard vaccine reduced virus titer by only 0.5-1 log. Thus, although the single dose regimen with the CCS-flu vaccine is inferior to the two-dose regimen for certain antibody isotypes, the two regimens provide a similar degree of protection.

In this experiment, we also compared CCS alone to CCS/Chol as the vaccine carrier (administered on days 0 and 7) and found no difference in immunogenicity between the two formulations (data not shown). Another formulation modification was the reduction of the size of the CCS/Chol lipid assemblies (diameter 0.05-5 μm) by extrusion (diameter ≦0.02 μm). Antibody titers induced by the extruded vaccine were 50-80% lower than those produced by the non-extruded vaccine (data not shown). Thus, unsized CCS lipid assemblies, with or without cholesterol, are highly efficient as a vaccine carrier for trivalent flu vaccine. TABLE 8 Elicitation of hemagglutination inhibition (HI) antibodies following intranasal vaccination with trivalent influenza vaccine, free and in CCS lipid assemblies, administered once or twice at various time intervals to young (2 mo.) BALB/C mice Mean HI titer (% seroconversion)^(b) Dosing A/New Caledonia A/Panama B/Yamanashi No. Vaccine^(a) (n = 5) days serum lung serum lung serum lung 1 None (PBS) ×2 0, 7 0 0 0 0 0 0 2 F-HN 2 μg × 1 0 0 0 0 0 0 0 3 4 μg × 1 0 0 0 0 0 0 0 4 2 μg × 2 0, 3 0 0 0 0 0 0 5 2 μg × 2 0, 7 0 0 0 0 0 0 6 2 μg × 2 0, 14 0 0 0 0 0 0 7 Lip (CCS/Chol)-HN 2 μg × 1 0 0 0 0 0 0 0 8 4 μg × 1 0 336 (100) 40 328 (100) 40 52 (80) 0 9 2 μg × 2 0, 3 544 (100) 80 408 (100) 40 52 (80) 0 10 2 μg × 2 0, 7 544 (100) 80 544 (100) 120 88 (100) 0 11 2 μg × 2 0, 14 480 (100) 60 368 (100) 40 80 (80) 0 12 F-HN + CT (1 μg) 2 μg × 2 0, 7 608 (100) 80 664 (100) 120 84 (80) 0 ^(a)Mice were immunized with Fluvirin ® 2003/2004 trivalent subunit vaccine preparation consisting of A/New Caledonia/20/99 (H1N1)-like, A/Moscow/10/99 (H3N2)-like and B/Hong Kong/330/2001-like, either free (F-HN) or incorporated into CCS/Chol (3/2 mole ratio) lipid assemblies (0.6 mg for groups 7, 9, 10, 11; 1.2 mg for group 8). ^(b)Serum HI titer was determined on individual mice 35 days after the first vaccine dose. Lung (pooled) HI titer was tested on day 42. In parentheses - % of mice with HI titer ≧40.0 denotes HI titer <20.

TABLE 9 Elicitation of serum, lung and nasal antigen-specific IgG1, IgG2a and IgA antibodies following intranasal vaccination with trivalent influenza vaccine, free and in CCS lipid assemblies, administered once or twice at various intervals to young (2 mo.) BALB/c mice Mean antibody titer Dosing Serum Lung Homogenate Nasal wash No. Vaccine^(a) (n = 5) days IgG1 IgG2a IgG1 IgG2a IgA IgG1 IgG2a IgA 1 None (PBS) ×2 0, 7 0 0 0 0 0 0 0 0 2 F-HN 2 μg × 1 0 0 0 0 0 0 0 0 0 3 4 μg × 1 0 320 90 1500 0 0 0 0 0 4 2 μg × 2 0, 3 0 0 0 0 0 0 0 0 5 2 μg × 2 0, 7 0 0 0 0 0 0 0 0 6 2 μg × 2 0, 14 40 0 0 0 0 0 0 0 7 Lip (CCS/Chol)-HN 2 μg × 1 0 300 0 600 0 0 0 0 0 8 4 μg × 1 0 12000 4500 13000 0 0 0 0 0 9 2 μg × 2 0, 3 15000 10000 15000 2500 3500 0 10 0 10 2 μg × 2 0, 7 15000 12000 14000 2500 9000 200 30 100 11 2 μg × 2 0, 14 13000 5500 12000 1800 3000 50 0 0 12 F-HN + CT (1 μg) 2 μg × 2 0, 7 21000 15000 20000 2500 2000 250 30 45 ^(a)See Table 8 for experimental details. Samples were pooled and tested by ELISA against the 3 viral strains (pooled HN) 42 days after the first vaccine dose. 0 denotes titer <10.

TABLE 10 Protection of young BALB/c mice against viral challenge following intranasal vaccination with trivalent influenza vaccine, free and in CCS lipid assemblies Dosing No. Vaccine^(a) (n = 5) days Lung virus titer (log 10)^(b) 1 None — 6 2 F-HN 4 μg × 1 0 5.5 3 F-HN 2 μg × 2 0, 7 5 4 Lip (CCS/Chol)-HN 4 μg × 1 0 0 5 Lip (CCS/Chol)-HN 2 μg × 2 0, 7 0 6 F-HN 2 μg + CT (1 μg) × 2 0, 7 0 ^(a)See table 8 for experimental details. In groups 4, 5 the lipid/HN w/w ratio was 100/1. ^(b)The mice were infected intranasally 42 days after the first vaccine dose, using ˜10⁶ egg infectious dose 50% (EID 50) of the mouse-adapted reassortant X-127 virus (A/Beijing/262/95 [H1N1] × X-31 [A/Hong Kong/1/68 × A/PR/8/34). Lungs were harvested 4 days later, homogenized, serially diluted, and injected into the allantoic sac of 10 d. fertilized chicken eggs. After 48 h at 37° C. and 16 h at 4° C., 0.1 mL of # allantoic fluid was removed and checked for viral presence by hemagglutination.

In the experiment described in Tables 11 and 12, the trivalent-flu vaccine was formulated with the CCS/Chol lipid assemblies using varying amounts of the HN antigens and the lipid. In this experiment the vaccines were prepared with: (a) varying amounts of the antigen (0.25-2 μg per viral strain) and of the lipid (0.075-0.6 mg), keeping the lipid/HN w/w ratio constant at 100/1; (b) graded amounts of the antigen (0.25-2 μg) and a constant amount of the lipid (0.6 mg) thereby varying the lipid/HN w/w ratio from 100/1 to 800/1. As can be seen in Table 11 (HI titer) and Table 12 (isotype titers) vaccines prepared at a 100/1 lipid/HN w/w ratio using 2 or 1 μg antigen of each strain and 0.6 or 0.3 mg lipid, respectively, produced high and similar levels of antibodies against the 3 viral strains (groups 2, 3). At lower antigen (0.5, 0.25 μg/strain) and lipid (0.15, 0.075 mg) doses the response decreased markedly (groups 4, 5), particularly the mucosal response (lung, nasal) (Table 12). When a constant dose of lipid was used (0.6 mg), high levels of antibodies were obtained even with the two lower doses of antigen (0.25, 0.5 μg/strain) (groups 6-8). Thus, the amount of the CCS lipid is critical, and with the appropriate lipid dose the antigen dose can be reduced 4-8 fold (from 1-2 μg to 0.25-0.5 μg) namely a clear dose-sparing effect. TABLE 11 Effect of the antigen dose and lipid dose on the induction of HI antibodies following intranasal vaccination with trivalent influenza vaccine formulated with CCS lipid assemblies, administered twice (at 1 week interval) to young (2 mo.) BALB/c mice Mean HI titer (% seroconversion) A/New Lipid/HN Caledonia A/Panama B/Yamanashi No. Vaccine^(a) (n = 5) HN (μg) Lipid (mg) w/w ratio Serum Lung Serum Lung Serum Lung 1 F-HN 2 — —  0 0  0 0  0 0 2 Lip (CCS/Chol)-HN 2 0.6 100/1 544 (100) 80 544 (100) 120  88 (100) 0 3 1 0.3 100/1 320 (100) 80 544 (100) 160  40 (100) 0 4 0.5 0.15 100/1 416 (100) 20 448 (100) 40  32 (100) 0 5 0.25 0.075 100/1 180 (100) 0 100 (100) 20  0 0 6 1 0.6 200/1 672 (100) 80 736 (100) 160 104 (100) 0 7 0.5 0.6 400/1 560 (100) 80 608 (100 160 104 (100) 0 8 0.25 0.6 800/1 512 (100) 80 512 (100) 120  48 (100) 0 ^(a)See Table 8 for experimental details.

TABLE 12 Effect of the antigen dose and lipid dose on the induction of serum, lung and nasal antigen-specific IgG1, IgG2a and IgA antibodies following intranasal vaccination with trivalent influenza vaccine formulated with CCS lipid assemblies, administered twice (at 1 week interval) to young BALB/c mice Lipid/HN Mean antibody titer Vaccine^(a) HN Lipid w/w Serum Lung homogenate Nasal wash (n = 5) (μg) (mg) ratio IgG1 IgG2a IgG1 IgG2a IgA IgG1 IgG2a IgA 1 F-HN 2 — — 0 0 0 0 0 0 0 0 2 Lip (CCS/Chol)-HN 2 0.6 100/1 15000 12000 14000 2500 9000 200 30 100 3 1 0.3 100/1 14000 2500 10000 1000 8000 100 0 80 4 0.5 0.15 100/1 15000 1300 8000 1500 4000 0 0 0 5 0.25 0.075 100/1 12000 400 3500 400 2500 0 0 0 6 1 0.6 200/1 20000 15000 12000 2500 8000 200 15 80 7 0.5 0.6 400/1 15000 14000 15000 5000 15000 150 35 100 8 0.25 0.6 800/1 15000 9000 21000 2500 13000 250 25 90 ^(a)See Tables 8, 9 for experimental details.

In a further experiment, the subunit flu vaccine, either free (HN) or associated with the CCS/Chol lipid assemblies (Lip HN), was tested for its ability to induce HI antibodies cross-reacting with various influenza A and B substrains that were not included in the vaccine. The data shown in Table 13 indicate that intranasal (i.n.) and intramuscular (i.m.) vaccination, administered once or twice, with either a monovalent or trivalent CCS-based influenza vaccine, elicits high serum titers of HI antibodies directed against the immunizing strains, as well as HI antibodies cross-reacting with several A/H1N1, A4H3N2 and B strains that were circulating in the years 1986-1999 and were not included in the vaccine. Slightly lower HI titer was found after a single i.n. vaccine dose (gr. 6 vs. gr. 7). Lung homogenate HI titers (gr. 4, 8) were lower than the corresponding serum titers. Thus, parenteral or intranasal vaccination with the CCS-based vaccine may afford protection against a wide spectrum of A and B viral strains. Such antigenic variants may emerge during a flu epidemic/pandemic as a result of antigenic drift. In contrast, the standard commercial vaccine administered i.n. (gr. 1, 5) was totally ineffective in inducing antibodies against both the homologous and the heterologous strains. TABLE 13 Induction of strain cross-reactive HI antibodies following intranasal or intramuscular vaccination of young BALB/c mice with CCS-based monovalent and trivalent influenza vaccine Mean HI titer against: A/H1N1 New A/H3N2 Cale- Tex- Singa- Pana- Johan- B Vaccine Sample donia/ Beijing/ as/ pore/ ma/ Sydney/ Nanchang/ nesburg/ Yamanashi/ Harbin/ No Vaccine^(a) strains tested 20/99 262/95 36/91 6/86 2007/99 5/97 333/95 33/94 166/98 07/94 1 HN A/New serum 0 0 0 0 0 0 0 0 0 0 2 μg × 2 i.n. Caledonia 2 Lip HN serum 1280 1280 1280 240 0 0 0 0 0 0 2 μg × 2 i.n. 3 Lip HN serum 640 640 320 40 0 0 0 0 0 0 1 μg × 1 i.m. 4 Lip HN lung 320 240 240 20 0 0 0 0 0 0 2 μg × 2 i.n. homogenate 5 HN A/New serum 0 0 0 0 0 0 0 0 0 0 2 μg × 2 i.n. Caledonia, 6 Lip HN A/Panama, serum 320 80 120 0 320 320 120 120 60 120 4 μg × 1 i.n. B/Hong 7 Lip HN Kong serum 480 120 240 20 640 640 120 120 80 320 2 μg × 2 i.n. 8 Lip HN lung 80 80 40 0 120 80 0 0 0 40 2 μg × 2 i.n. homogenate 9 HN 2 μg + serum 480 240 120 40 480 480 120 120 80 240 CT 1 μg × 2 i.n. ^(a)Pooled sera and lung homogenate obtained 5 weeks after vaccination were tested for HI antibodies. For experimental details, see Table 8. The lipid (Lip) assemblies were composed of CCS/Chol (3/2 mole ratio) and the lipid/HN w/w ratio was 300/1 in groups 2-4 and 100/1 in groups 6-8. Except for groups 3 and 6, the two vaccine doses were spaced 1 week apart. In bold, antibody titers against the immunizing strains. 0 denotes HI titer <10. Biodistribution of Anionic and Cationic Liposomes Loaded with HN and Administered Intranasally

In a biodistribution experiment, 3 formulations of lipid assemblies: DMPC/DMPG (anionic), DOTAP/Chol (cationic) and CCS/Chol (cationic), either empty or loaded with the influenza HN antigens, were administered intranasally (200 kg lipid, 2 μg antigen per mouse) into BALB/c mice. The fluorescently labeled lipid was then traced in the homogenates of various tissues (nose, lungs, gastrointestinal tract, brain, liver, kidneys, heart and spleen) over a period of 24 h (at 1, 5, and 24 hours post administration).

As can be seen in the following Table 14 and in FIG. 2A-2F, after 1 and 5 hours there was 75-100% recovery of total lipid administered (% from the administered dose) of all the three formulations tested. This recovery however dropped significantly at 24 hours in all formulations except for the CCS formulation. The CCS formulation containing the HN antigens displayed the longest retention (>24h.) in the 3 target organs (nose, lungs, GI tract) while there was no lipid accumulation in the brain and no significant accumulation in the other organs tested (liver, kidneys, heart, spleen). TABLE 14 Recovery at 1, 5, and 24 hours of fluorescently labeled lipid assemblies administered intranasal % Recovery (of total lipid administered) in nose, lungs, gastrointestinal tract, brain, liver, kidneys, heart and spleen Lipid assembly formulation 1 hour 5 hours 24 hours DMPC/DMPG (empty) 100.2 99.3 26.9 DMPC/DMPG:HN 100.2 99.9 8.3 DOTAP/Chol (empty) 107.0 75.1 8.1 DOTAP/Chol:HN 99.9 106.4 6.7 CCS/Chol (empty) 99.6 96.9 74.2 CCS/Chol:HN 101.1 101.5 94.5 *The values show % recovery of the lipid as related to total dose administered.

When ¹²⁵I-labeled HN was used, its biodistribution resembled that of the fluorescent lipid. This long retention of the CCS vaccine components in the respiratory and GI tracts may explain, in part, its superior immunogenicity over the other liposomal formulations. This is exhibited in the following study in which the antigen component of the vaccine was traced. HN proteins were labeled with ¹²⁵I and administered intranasally either free or associated with one of the lipid formulations used in the fluorescent biodistribution experiments. Radioactivity of the various tissues was determined at 1, 5 and 24 h post instillation.

Table 15 teaches that recovery of the antigen was high in this experiment as well. As can be seen in FIGS. 3A-3D, the biodistribution pattern of the ¹²⁵I-labeled HN is similar to that of the lipid (FIGS. 2A-2F), further establishing that: (a) there is indeed an in vivo association between the HN-proteins and the lipid assemblies, and (b) the prolonged retention in the nose of the antigen when associated with the cationic lipid assemblies may be due to the cationic lipid assemblies and not an inherent property of the HN proteins, since there is no HN retention when the protein is administered by itself in soluble form.

Also this experiment may teach that there is no HN protein accumulation in the brain when administered alone or associated with lipid-assemblies (a major safety concern with intranasal vaccination). Since the radioactive tracing method is much more sensitive than the fluorescent method, this result is more confidently based. TABLE 15 Recovery of ¹²⁵I labeled HN administered intranasally either alone or associated with lipid assemblies at 1, 5 and 24 hours Recovery (% of total administered) nose, lungs, gastrointestinal tract, brain, liver, kidneys, Lipid heart and spleen) assembly formulation 1 hour 5 hours 24 hours HN 77 48 17 Lip (DMPC:DMPG) HN 88 50 26 Lip (DOTAP:Chol) HN 105 58 32 Lip (CCS:Chol) HN 100 74 41 *The values show % recovery of the HN antigen as related to total dose administered.

In an attempt to test if the protein and lipid are retained and/or cleared by similar or different kinetics in the various tissues, another analysis of the data was performed, where the ratio between the % antigen retention (of the total dose administered) and % lipid retention in the various tissues at various time points was determined. When the ratio is constant and ≅1, it means that both components were similarly retained in the same organ, while when this ratio is either larger or smaller than 1 it suggests that the clearance kinetics of each component was different, and one component was cleared faster than the other.

As can be seen in Table 16 below, the only ratio that remained constant with time was that of CCS/Chol-HN in the nose (ratio=˜0.45). This suggests that: (a) the high retention of the antigen in the nose with CCS and DOTAP is in correlation with the level of association and due to the binding of these formulations to the nasal mucosa, in contrast to DMPC/DMPG; and (b) while the other formulations' components dissociate in the body and are cleared at different rates, the CCS-HN based formulation was stable, especially in the nose, and this may contribute to the enhanced immunogenicity seen with the CCS-based vaccines. TABLE 16 Time-dependent biodistribution of vaccine component (lipid and HN antigen) after i.n. administration Lip (DMPC:DMPG) Lip (DOTAP:Chol) HN HN Lip (CCS:Chol) HN 1 h 5 h 24 h 1 h 5 h 24 h 1 h 5 h 24 h HN nose  9%  4% 2% 38% 16% 2% 41% 14% 12% lungs 30%  3% 4% 19%  4% 12%  24% 21% 11% GI 35% 32% 11%  33% 27% 10%  22% 28%  8% recovery: 88% 50% 26%  105%  58% 32%  100%  74% 41% lipid nose  0%  0% 0% 46% 56% 0% 88% 30% 25% lungs 67% 80% 5% 38%  3% 3% 12% 35% 14% GI 33% 20% 4% 16% 47% 3%  1% 37% 55% recovery: 100%  100%  8% 100%  106%  7% 101%  102%  95% HN/lipid nose — — — 0.82 0.29 — 0.47 0.45 0.47 ratio lungs 0.44 0.03 0.85 0.50 1.29 3.56 2.01 0.60 0.80 GI 1.07 1.56 2.96 2.12 0.57 2.86 16.08 0.76 0.15 recovery: 0.88 0.50 3.12 1.05 0.55 4.78 0.99 0.73 0.44 The values show % recovery of the lipid or HN protein as related to total dose administered. Preliminary Safety Study of the Intranasal Flu Vaccine

Toxicity (local, systemic) is a major concern with both i.m. and i.n. vaccines and therefore a pilot toxicity study was studied. Cationic lipid formulations (DMTAP, DOTAP, CCS-based) loaded with the influenza antigens hemagglutinin+neuraminidase (HN) were administered i.n. (twice, spaced 1 week apart) to mice (n=4/group), and blood counts (total, differential), blood chemistry and histological examination (nose, lung sections) were performed 72 hours later. The mice showed no apparent signs of any toxicity. Blood counts and blood chemistry were within the normal range, and, as expected, minimal-mild inflammatory response was seen in the nose and lungs of mice treated with the cationic formulations. A similar, albeit less pronounced, inflammatory response was also seen in some mice treated with saline alone or with the non-encapsulated antigen.

Immunomodulatory Activity of CCS-Flu Vaccine in Mice

In these experiments, mice were injected i.p. with various liposomal formulations (composed of DMPC, DMPC/DMPG, DOTAP/Chol, CCS/Chol), 0.5-1 mg lipid, with or without the HN antigens. The mice were either untreated or i.p. injected with thioglycollate (TG, to increase macrophage production) 2 days before the injection of the liposomal formulations. Peritoneal cells were harvested 24-48 h. after administration of the liposomes and used as such or after 4 h. adsorption at 37° C. to plastic dishes and removal of the non-adherent cells. In other experiments, peritoneal cells were harvested from TG treated mice and incubated with the liposomal formulations for 24-48 h. The cells were tested by flow cytometry for the expression of MHC II and the co-stimulatory molecules CD40 and B7. The supernatants were tested for the cytokines interferon γ (IFN γ), tumor necrosis factor α (TNF α) and interleukin 12 (IL-12), and for nitric oxide (NO).

All the cationic formulations (CCS/Chol, DOTAP/Chol, DMTAP/Chol) upregulated the expression of B7 and CD40 more than the other formulations (DMPC [neutral], DMPC/DMPG [anionic]) and induced higher levels of IFN γ and IL-12. In some cases the CCS/Chol formulation was more effective than the other cationic formulations. No significant levels of TNF α and NO were induced by any of the formulations. The enhanced expression of co-stimulatory molecules on antigen presenting cells and the induction of IL-12 and IFNγ by the cationic formulations can explain, in part, the greater adjuvant activity of these formulations. These findings combined with the long retention of the CCS-flu vaccine in the respiratory tract (FIGS. 2C and 2F and FIGS. 3A-3D) after intranasal administration may explain why CCS is such an efficient mucosal vaccine carrier/adjuvant.

Immunogenicity of Isolated CCS Isomers or their Synthetic Mixture (CCS)

As indicated above, in order to assess the immunogenicity of the isolated CCS isomers, i.e. N-palmitoyl D-erythro sphingosyl-1-carbamoyl spermine (C-1 CCS) or (N-palmitoyl D-erythro sphingosyl-3-carbamoyl spermine (C-3 CCS), the following different formulations were evaluated:

CCS/C-Ag: a formulation of CCS, cholesterol and the antigen, where the CCS is obtained by the synthetic procedure described above;

C-1-CCS/C-Ag: a formulation of the isolated C-1 CCS isomer, cholesterol and the antigen;

C-3-CCS/C-Ag: a formulation of the isolated C-3 CCS isomer, cholesterol and the antigen;

C-1-CCS/C-3-CCS/C-Ag: a formulation of a mixture of the isolated C-1 CCS isomer, C-3 CCS isomer (mole:mole ratio of 80:20), cholesterol and the antigen.

As control, free Ag was used (F-Ag).

The anti A/New Caledonia titers were determined and are presented in Table 17: TABLE 17 Serum HI titers 4 weeks post immunization (i.m. administration) anti A/New Caledonia titers Vaccine average SD Median Free-Ag 70 30 80 Lipids prepared by the dichloromethane/methanol method CCS/C-Ag 1550 870 1280 C-1 CCS/C-Ag 1040 880 960 C-3 CCS/C-Ag 2240 1610 1920 C-1CCS/C-3 CCS/C-Ag 590 260 640 Lipids prepared by DDW/T-Butanol and lyophilization method CCS/C-Ag 910 710 960 C-1 CCS/C-Ag 560 130 640 C-3 CCS/C-Ag 2190 1900 1600 C-1CCS/C-3 CCS/C-Ag 1000 830 640

The results presented in Table 17 show that both C-1 and C-3 CCS isomers effectively enhance the response to the antigen, with a significantly greater effect exhibited when using as an adjuvant the isolated C-3 CCS isomer as compared to the C-1 isomer.

Immunogenicity of CCS in Rat Model

The HI antibody levels of CCS/C-HA vaccinated rats was significantly higher than that of mice vaccinated with the commercial vaccine alone (p<0.05) at all tested time points as shown in FIG. 4.

Protective Efficacy of CCS/C in Ferret Model

FIG. 5 shows the mean sum of virus titer in nasal wash following infection of ferrets with the antigen. The results show that the CCS/C vaccinated group, both the 15 kg and 5 μg HA dose, had significant reduction in mean sum virus titer following infection as compared to the CCS/C only group and also as compared to Free-HA group (P<0.001).

Avian Influenza Vaccine

The feasibility of improving the anti-influenza vaccine, as compared to an Alum-adsorbed Ag, by using a single dose of inactivated whole virus with the polycationic sphingolipid, CCS, in mice was also examined by determining serum anti-hemagglutinin (HA) antibodies.

Materials and Methods

Antigen (Ag): H5N1—A/Vietnam/1194/2004, inactivated purified whole virus (NIBSC, Hertforshire, UK). ˜60 μg/ml HA, ˜160 μg/ml total protein.

Mice: BALB/C females aged 9 weeks, n=5-6/group.

Treatments

Doses: a single-dose vaccination as follows:

Free Ag: 3 or 6 μg per animal

Liposomal formulation: The liposomes were composed of CCS and Cholesterol (CCS/C, [Biolab, Jerusalem] and Cholesterol [Minakem, France]) at mole:mole ratio of 3:2. lipid:HA w/w ratio 250: 1, lipid:total protein w/w ratio 75:1.

Alum-Ag: Aluminium hydroxide (Alhydrogel 2%, Sigma Israel). Alum:HA 25:1 w/w. Alum adsorption: 2 hours at RT with shaking.

Administration: intramuscularly (i.m.) into the hind leg, 50 μl to one leg for 3 μg and 50 μl/leg to 2 legs for 6 μg. TABLE 18 Treatment group assignment for animals Group Formulation Dose administration number 1 F—Ag 2 Alum-Ag 3 μg HA + 0.75 6 mg lipid 3 CCS/C-Ag i.m. × 1 4 F—Ag 5 5 Alum-Ag 6 μg + 1.5 mg lipid 6 6 CCS/C-Ag 6

Serum samples were collected at weeks 2, 4, 8, 12 and 20 after immunization.

Antibody level determination: Serum anti-hemagglutinin titer was determined by hemagglutination inhibition assay (HI).

Statistical analysis: The difference between the test groups was done by student t-test.

Results

The results are presented in FIG. 6. Specifically shown are the serum HI Ab titers against H5N1 virus (A/Vietnam/1194/2004) with the different agents: free Antigen (F-Ag); Alum-antigen (alum-Ag); and CCS/cholesterol-Antigen (CCS/C-Ag), at two different Ag dose levels (3 μg and 6 μg). It is clear that when using liposomal CCS/cholesterol as the carrier, a much higher HI antibody titer is obtained (*p=0.04 as compared to Alum-Ag i.m. 3 μg at 8 weeks).

Hepatitis A Virus (HAV)

In addition to influenza, the immune enhancing potential of CCS lipid assemblies was also tested for HAV vaccine administered by the intranasal (i.n.) and the intrarectal (i.r.) routes in BALB/C mice.

HAV vaccine (Aventis Pasteur), 10 EU (˜1.5 μg protein), was administered twice at a 2-week interval and the response was tested by the ELISPOT technique 3 weeks after the second vaccine dose. CpG-ODN, used as a mucosal adjuvant, was given at 10 μg/dose. The HAV-CCS lipid assemblies were prepared as described above for the influenza vaccine (Table 1).

The data presented in Table 19 show that whereas the commercial HAV vaccine failed to induce an IgA response in both tissues (lamina propria, Peyer's patches) tested, and by both administration routes (i.n., i.r.), the vaccine formulated with either CCS or CpG-ODN generated a significant response in most cases. The combination of HAV-CCS lipid assemblies and CpG-ODN resulted in a synergistic response in all cases. Thus, CCS lipid assemblies alone, and particularly in combination with CpG-ODN, are also effective as a carrier/adjuvant for mucosal vaccination against HAV. TABLE 19 Induction of IgA antibodies following intranasal (i.n.) or intrarectal (i.r.) vaccination of BALB/c mice with hepatitis A virus (HAV) vaccine, alone and in combination with CCS lipid assemblies and/or CpG-ODN Mean no. of IgA AFC^(a)/ 10⁶ cells in: Lamina Peyer's propria patches Vaccine i.n. i.r. i.n. i.r. HAV alone 0 0 0 0 HAV-CCS 12 27 0 1 HAV + CpG-ODN 16 22 0 14 HAV-CCS + CpG-ODN 139 68 28 23 ^(a)AFC—antibody-forming-cells C. Botulinum

In a further experiment, Mice were immunized i.n. with 0.4 μg dose of a commercial C. botulinum toxoid (CBT, as a model for bioterror agent, Uruguay, alum free) and antibody titers were tested by ELISA 4 weeks after the second vaccine dose.

The results of an experiment with C. botulinum toxoid are summarized in Table 20, which shows the superiority of the CCS-toxoid formulation over the standard vaccine following i.n. instillation, particularly with regard to the TgA levels in the small intestine and feces. Such Antibodies are expected to neutralize the toxin upon oral exposure. Mice immunized i.n. with the vaccine alone did not produce IgA. TABLE 20 Induction of IgG1, IgG2a and IgA antibodies in BALB/c mice vaccinated intranasally (twice, 1 week apart) with free or CCS-associated Clostridium botulinum toxoid (CBT) Mean antibody titer Vaccine^(a) Serum Small intestine Feces n = 10 IgG1 IgG2a IgG1 IgG2a IgA IgA CBT 0 0 1000 180 0 0 CCS-CBT 400 24 1600 0 1800 1800 Hepatitis B Virus (HBV)

In addition, the immune enhancing potential of CCS lipid assemblies was also tested for vaccination of mice against Hepatitis B.

Hepatitis B surface antigen particles derived from CHO cells were characterized as described before [Diminsky, D., et al. “Comparison between hepatitis B surface antigen (HBsAg) particles derived from mammalian cells (CHO) and yeast cells (Hansenula polymorpha): composition, structure and immunogenicity” Vaccine 15:637-647 (1997); Diminsky, D., et al. “Physical, chemical and immunological stability of CHO-derived hepatitis B surface antigen (HBsAg) particles”. Vaccine 18:3-17 (1999)]. In addition, the electrostatic properties of the recombinant HBsAg particles were characterized for their Zeta Potential using Zetasizer 3000 HAS, Malvern Instruments, Malvern, UK, and as also described by Garbuzenko O. et al. [Garbuzenko O. et al. “Electrostatics of PEGylated micelles and liposomes containing charged and neutral lipopolymers” Langmuir 21:2560-8 (2005)]. HBsAg particles has a negative zeta potential −26.7 mV. This suggests that these particles bind with cationic CCS/Chol assemblies.

Experiment 1 : Intraperitoneal and Intranasal Vaccination

The liposomal formulation was prepared at a lipid/protein antigen w/w ratio of 600/1 (CCS/Chol mole:mole ratio of 3:2). BALB/C mice (females, 8 weeks old, n=5-6) were vaccinated once i.p. or i.n. with purified CHO-derived recombinant HBsAg particles (S+preS1+preS2 particles), Scigen, Yavne, Israel) either alone (referred to as naked particles, F-Ag) or with CCS/Chol liposomal formulation and serum antibody levels were determined 5 and 12 weeks following vaccination. The vaccine dose was 1 μg HBsAg protein with 0.6 mg lipid for i.p. administration and 2 μg HBsAg protein with 1.2 mg lipid for i.n. administration.

The vaccination results are presented in FIGS. 7A-7B. Specific antibodies against HBsAg were detected by Microparticle Enzyme Immunoassay (MEIA Diminsky, D., et al. (1997) ibid.; Diminsky, D., et al. (1999) ibid.]). Specifically, FIG. 7A shows that vaccination with the liposomal CCS/Chol formulation resulted in higher serum levels of anti-HBsAg antibodies as compared to the naked antigen following both i.p. and i.n. administration.

Moreover, measurement of specific isotypes against HBsAg showed that only mice immunized with the CCS/Chol vaccine, both i.p. or i.n., produced IgG2a antibodies while those immunized with naked HBsAg did not (FIG. 7B).

Experiment 2: Intraperitoneal Vaccination

Materials and Methods

Antigens

Ag: Scigen HBsAg (S+preS1+preS2) without Alum (Scigen, Yavne, Israel) batch 6P-01-001-09.

Positive control: Sci-B-Vac™, Lot 03870101, (Scigen Ltd, Singapore), manufactured by BTG Israel, Alum-adsorbed Ag.

Animals: BALB/C mice, females aged 8 weeks, n=8 animals per group.

Treatment Schedule

Doses: a single-dose vaccination as follows:

Free Ag: 0.5 μg per animal

Liposomal formulation: The liposomes were composed of CCS and Cholesterol (CCS/C, batch no. NV/010905, MediWound, Yavne) at mole:mole ratio of 3:2. Lipid:Antigen weight ratio 1200:1. Therefore, 0.5 μAg with 0.6 mg total lipid per animal.

Sci-B-Vac™: 0.5 μg per animal

Administration: intraperitoneal (i.p.) 100 μl. TABLE 21 Treatment group assignment for animals Formulation dose administration 1 Scigen F-HBsAg 0.5 μg Ag 2 Sci-B-Vac ™ 0.5 μg Ag + 75 μg Alum i.p. × 1 3 Scigen CCS/C-HBsAg 0.5 μg Ag + 0.6 mg lipid

Blood collection for antibody detection: Blood was collected 14, 28 days, 2 months and 3 months post-vaccination, from the orbital sinus vein.

Antibody level determination: Serum total anti-HBsAg titer was determined by the AxSYM AUSAB MEIA (Abbot). IgG1, IgG2a were determined by ELISA.

Statistical analysis: The difference between mice vaccinated with Sci-B-Vac™ and CCS/C-Ag was done by Student t-test.

Results

The results are presented in FIGS. 8A-8C.

Specifically, FIG. 8A shows that a statistically significant higher anti-HBsAg titer is obtained with the composition comprising the antigen carried by liposomes composed of CCS in combination with cholesterol (*p<0.05 between CCS/C-Ag and Sci-B-Vac 8 weeks post-vaccination).

Further, FIG. 8B shows that a statistically significant higher anti-HBsAg IgG1 titer is obtained with the composition comprising the antigen carried by liposomes composed of CCS in combination with cholesterol (*p<0.05 between CCS/C-Ag and Sci-B-Vaccine at 4, 8 and 12 weeks post-vaccination, 0 denotes <40).

Yet further, FIG. 8C shows that a statistically significant higher anti-HBsAg IgG2a titer is obtained with the composition comprising the antigen carried by liposomes composed of CCS in combination with cholesterol (**p<0.01 between CCS/C-Ag and Sci-B-Vaccine, at 4, 8 and 12 weeks post-vaccination 0 denotes <40).

Anthrax

The immunogenicity of CCS/C-Anthrax vaccine was also examined in a mouse model (A) and in a guinea pig model (B). Specifically, the human anti-Bacillus anthracis vaccine comprising the bacterium's Protective Antigen (PA) was used to assess the feasibility of improving the anti-B. anthracis vaccine by using a single or double vaccination with the commercial PA in combination with CCS-based liposomes.

(A) Mouse Model

Materials and Methods

Ag: recombinant Anthrax Protective Antigen (PA) from B. anthracis List biological laboratories (INC, California, USA), lot no. 17111A1B.

Animals: BALB/C mice, females aged 8-9 weeks, n=4-6 per group.

Treatments

Doses: a single or double-dose vaccination as follows:

Free Ag: 10 μg or 20 μg per animal

Alum-Ag: Alhydrogel 2% (Sigma). Alum adsorption: 2 hours at RT with shaking. Two Alum:Ag w/w ratios: 7:1 and 25:1.

Liposomal formulation: The liposomes were composed of CCS and Cholesterol (CCS/C, batch no. NV/010905 [MediWound, Yavne]) at molar ratio of 3:2. Lipid:Antigen weight ratio 150:1 for s.c, 113:1 for i.n.

Administration: single subcutaneous (s.c) administration: 50 μl for 10 μg PA, 100 μl for 20 μg PA.

Double intranasal (i.n) administration: 30 μl, 20 μg per dose, at weeks 0 and 5. TABLE 22 Treatment group assignment for animals admin- Group Formulation Dose istration number 1 Free-Ag 10 μg Ag 4 2 Free-Ag 20 μg Ag 4 3 Alum-Ag 10 μg Ag + 70 μg Alum 6 7:1 (w/w) 4 Alum-Ag 10 μg Ag + 250 μg Alum s.c. × 1 6 25:1 (w/w) 5 Alum-Ag 20 μg Ag + 500 μg Alum 6 25:1 (w/w) 6 CCS/C-Ag 10 μg Ag + 1.5 mg Lipid 6 7 CCS/C-Ag 20 μg Ag + 3 mg Lipid 6 8 CCS/C-Ag 20 μg Ag + 2.25 mg i.n × 2 6 Lipid (0 and 5 w)

Blood collection for antibody detection: Blood was collected from the orbital sinus vein at weeks 2, 4 (for s.c. and i.n.), and 7 (only for i.n.) post-vaccination.

Antibody level determination: Individual serum samples analyzed for anti-PA antibodies titers by ELISA using a commercial kit: QuickELISA™ Anthrax-PA kit (Immunetics, Inc, MA, USA) (Veterinary Institute).

Results

The results are presented in FIGS. 9A-9B.

Specifically, FIG. 9A shows anti-PA IgG antibodies median levels (data presented as O.D) as determined using a commercial kit: QuickELISA™ Anthrax-PA kit (Immunetics, Inc, MA, USA). O.D>0.186 is considered as positive response. FIG. 9B shows the anti-PA antibodies median levels determined by ELISA. %—percentage of responders.

As shown in FIG. 9A, serum anti-PA levels (represented by their respective O.D.) and percentage of responding mice, 2 and 4 weeks post a single s.c. vaccination with the PA+CCS/C liposomes are much higher than those exhibited with Alum-Ag or the free antigen vaccination. A single i.n. dose of CCS/C-PA did not induce a significant antibody development. However, a second i.n. dose with CCS/C-PA on week 5 induced a robust antibody development 2 weeks later (FIG. 9B).

(B) Guinea Pig Model

Materials and Methods:

Antigens

PA: A commercial preparation of a recombinant anthrax protective antigen (List Biological laboratories, INC, California, USA) was used.

Positive control: Sterne strain (STI) live attenuated vaccine (Onderstepoort, Namibia), commonly used in veterinary medicine.

Animals: Guinea pigs are the laboratory animals most commonly used in experiments with B. anthracis. Hartley strain guinea pigs, weighing about 300 grams each.

Treatments:

Doses: a single-dose vaccination as follows:

Free PA: 25 μg

Liposomal formulation: The liposomes were composed of CCS and Cholesterol (CCS/C, batch MediWound #NV/010905) at mole:mole ratio of 3:2. Lipid:Antigen weight ratio 150:1. Therefore, 25 μg PA with 3.75 mg total lipid per animal

STI vaccine: 0.5 ml per animal

Administration: PA vaccine: subcutaneous (s.c) 100 μl, or STI vaccine: s.c. 0.5 ml. TABLE 23 Treatment group assignment for animals Group Formulation dose Administration 1 Free-Ag 25 μg Ag 2 CCS/C-Ag 25 μg Ag + 3.75 mg s.c. × 1 Lipid 3 Sterne strain (STI) 0.5 ml virus

Blood collection for antibody detection: Blood was collected 28 days post-vaccination, from the heart.

Antibody level determination: by Immunetics® QuickELISA™ Anthrax-PA kit (Immunetics, Inc, MA, USA).

Statistical analysis: The difference between guinea pigs vaccinated with PA and CCS/C-PA subcutaneously was done by the Kruskal-Wallis non-parametric ANOVA test (Statistix® package, version 7 (Analytical Software, USA).

Results

The results are presented in FIG. 10. Specifically, the figure shows anti-PA antibodies median levels (presented as O.D) in Guinea pigs as determined by a commercial kit: QuickELISA™ Anthrax-PA kit (Immunetics, Inc, MA, USA) (O.D>0.186 is considered as positive response) and that the anti-PA antibody levels are statistically higher with CCS/C-PA vaccine (* Significant statistical difference between the groups of CCS/C-PA vaccine and F-PA vaccine (p<0.001)).

Streptococcus pneumoniae

Proposed Preclinical Studies

Intranasal Challenge of Mice Immunized with rPsaA/CCS

Groups of 10 mice 3- to 5-week-old CBA/NCAHNXID mice (Jackson Laboratories, Barr Harbor, Me.) will be immunized intranasally (i.n.) using purified rPsaA at the following doses: 150 ng or 500 ng per animal each dose adjuvated with CCS (at an antigen:CCS ratio of between 1:10 to 1:600). For intranasal administration, 10 μl of each dose with or without CCS will be prepared freshly with 0.85% physiological saline using a stock solution of 1 mg/ml of purified rPsaA. Mice will be boosted twice with the same dose of rPsaA on day 7 and day 14 post-initial dose.

On day 14 following the final booster, saliva (60 μl/mouse) and blood (100 μl) from the tail vein will be collected from each mouse and analyzed for an IgG response by ELISA. Six weeks after final boosting (day 38), mice will be challenged with 10⁶ colony forming units of PLN D39 suspended in 10 μl of 0.85% saline. On day 7 post-challenge, mice will be euthanized and intranasal wash (6 drops) and blood (100 μl) will be collected. Both blood and intranasal wash samples will be serially diluted and plated to 5% sheep blood agar plate supplemented with 0.3 μg/ml erythromycin. Bacterial cultures will be incubated at 37° C. in a 5% CO₂ incubator, and colonies will be counted following 24 h incubation [De B. K. et al. Purification and characterization of Streptococcus pneumoniae palmitoylated pneumococcal surface adhesin A expressed in Escherichia coli. Vaccine; (2000) 18(17):1811-1821]

Immunization of Mice with Recombinant 6PGD and CCS

Six-week-old BALB/c female mice (Harlan Laboratories, Israel) will be immunized intraperitoneally with 25 μg of r6PGD and adjuvanted with 75 μl of CCS (at an antigen:CCS ratio of between 1:10 to 1:600) on days 0 (primary immunization), 7, 14 and 21 (booster). Control mice were sham immunized with CCS adjuvant only and another control group with r6PGD adsorbed to Alum. Blood samples will be collected from mice 1 week prior to immunization and 1 week after booster immunization. The sera will be pooled for immunological assays. For inhibition of adhesion experiments S. pneumoniae (10⁶ CFU) will be added to A549 cells, as described [D. Danieliy et al. Pneumococcal 6-phosphogluconate-dehydrogenase, a putative adhesin, induces protective immune response in mice. Clinical & Experimental Immunology Volume 144 Issue 2 Page 254—(2006)] prior to or after 30 min incubation with serum obtained from r6PGD immunized mice. The bacteria will be spun and resuspended in culture media prior to their addition to the cultured A549 cells. Incubation and results evaluation will be performed, again, as described by D. Danieliy et al. [Clinical & Experimental Immunology (2006) ibid.].

Respiratory Challenge with S. pneumoniae Strain WU2

For respiratory challenge r6PGD/CCS immunized (n=29) and control (n=14) mice will be anaesthetized and inoculated intranasally with 1×10⁸ CFU of S. pneumoniae strain WU2 (in 25 μl PBS). This inoculum's size is used as it was found to be the lowest that causes 100% mortality in our mouse model system within 96 h. Survival was monitored daily. The experiments will be conducted on three different occasions and the results will be pooled. Bacterial load will be determined in r6PGD/CCS immunized (n=3) and control mice (n=3). The nasopharynx and lungs will be excised and homogenized and blood will be withdrawn. Bacterial load in the nasopharynx, lungs and the blood is determined 48 h following intranasal challenge with S. pneumoniae strain WU2.

In a similar assay, immunization and challenge will be performed using CW-T, CW-L or CW-NL as antigens [M. Portnoi et al. The vaccine potential of Streptococcus pneumoniae surface lectin- and non-lectin proteins. Vaccine 24 (2006) 1868-1873] in combination with CCS at different antigen:adjuvant ratios between 1:10 to 1:600. Specifically, six week old BALB/c and C57BL/6 female mice; Harlan Laboratories, Israel) will be intramuscularly immunized with 25 μg of CW-T, CW-L or CW-NL adjuvanted with CCS at day 0 (primary immunization) and CCS (about 1.5 mg), days 7 and 14 (booster immunizations). Control mice will be sham immunized with CCS adjuvant alone and another control groups with CW-T, CW-L or CW-NL adsorbed to Alum. Mice will be bled 1 week prior to immunization and 1 week after second booster. The sera will be pooled. For challenge, with virulent S. pneumoniae strain WU2 (LD50 for intranasally challenge was 1×10⁷ CFU and for intraperitoneally challenge was 1×10⁶ CFU), mice were anaesthetized with pentobarbital sodium (0.6 mg/kg), and inoculated intranasally with 5×10⁸ CFU of S. pneumoniae (in 25 μl PBS) or intraperitoneally with 5×10⁷ CFU of S. pneumoniae (in 100 μl PBS). Mortality will be monitored daily.

Proposed Clinical Trials

Immunization of Human Subjects with Recombinant Truncated Rx1 Strain rPspA in Combination with CCS

Human Study

Patients aged 18±45 are immunized with recombinant truncated Rx1 strain rPspA (1±314) [G. S. Nabors et al. Vaccine 18 (2000) 1743-1754] adjuvanted with CCS prepared as described above. Groups of 30 adults will be immunized with rRx1 alone or with CCS (at a rRx1:CCS ratio of between 1:10 to 1:600) on day 0, Day 7 and day 14, and sera will be collected at various times after immunization. The control group will include immunization with Alum (ahydrogell Al(OH)₃ gel, superfos biosector a/s Denmark).

Human sera will be assayed for their ability to bind to rPspAs representing each of the six recognized clades using ELISAs. For each assay, the ELISA method will be essentially the same. Briefly, plates will be coated overnight at 48° C. with 0.5±1 mg/ml rPspA antigen in a volume of 100 ml/well. Plates will be washed with PBS containing 0.05% Tween-20, and test sera will be serially diluted across plates. After a 2 h incubation at room temperature, plates will be washed, and a 1:500 dilution of goat anti-human IgG Fc (Kirkegaard and Perry, Gaithersburg, Md.) will be added.

The invention will now be defined by the appended claims, the contents of which are to be read as included within the disclosure of the specification. 

1. A method for stimulating or enhancing an immune response of a subject to provide protection against an infection, the method comprising administering to said subject a combination of sphingoid-polyalkylamine conjugate and a biologically active molecule, the combination being effective to provide said stimulation or enhancement of the immune response, wherein said infection is caused by an agent selected from the group consisting of hepatitis B virus (HBV), avian influenza virus (AIV), the bacterium Bacillus anthracis and the bacterium Streptococcus pneumoniae.
 2. The method of claim 1, wherein said sphingoid-polyalkylamine conjugate comprises a sphingoid backbone carrying, via a carbamoyl linkage, at least one polyalkylamine chain.
 3. The method of claim 1, wherein said biologically active molecule is associated with said sphingoid-polyalkylamine conjugate.
 4. The method claim 1, wherein said biologically active molecule is selected from an antigenic protein, antigenic peptide, antigenic polypeptide, carbohydrate, or antigenic glyco-protein.
 5. The method claim 1, wherein: (i) when said infection is caused by HBV, said biologically active molecule is an Hepatitis B antigen; (ii) when said infection is caused by avian influenza virus, said biologically active molecule is an AIV antigen; (iii) when said infection is caused by the bacterium Bacillus anthracis, said biologically active molecule is an antigen of said bacterium; or (iv) when said infection is caused by the bacterium Streptococcus pneumoniae said biologically active molecule is an antigen of said bacterium.
 6. The method of claim 5, wherein: (i) said HBV antigen is an HBsAg particle; (ii) said AIV antigen is an inactivated purified whole avian influenza virus; (iii) said bacterium antigen is the protective antigen (PA) moiety of anthrax toxin; or (iv) said Streptococcus pneumoniae antigen comprises a polysaccharide or protein conjugate of an antigen selected from pneumococcal surface protein A (PspA), pneumococcal adherence and virulence factor A (PavA), pneumococcal glutamyl-tRNA synthetase, pneumolysin, cholin binding protein A (CbpA), pneumococcal surface adhesion A (PsaA).
 7. The method of claim 1, further comprising administering to said subject an immunostimulating agent.
 8. The method of claim 1, wherein said sphingoid-polyalkylamine conjugate forms a lipid assembly.
 9. The method of claim 1, wherein said sphingoid is ceramide.
 10. The method of claim 1, wherein said polyalkylamine is selected from spermine, spermidine, a polyamine analog or a combination of same thereof.
 11. The method of claim 1, wherein said sphingoid-polyalkylamine conjugate is N-palmitoyl-D-erythro-sphingosyl carbamoyl-spermine (CCS).
 12. The method of claim 1, wherein said sphingoid-polyalkylamine conjugate has the following formula (I):

wherein R₁ represents a hydrogen, a branched or linear alkyl, saturated or unsaturated cycloalkyl, aryl, hydroxyalkyl, alkylamine, or a group —C(O)R₅; R₂ and R₅ represent, independently, a branched or linear C₁₀-C₂₄ alkyl, hydroxyalkyl, alkenyl, saturated or unsaturated cycloalkyl, aryl or polyenyl groups; R₃ and R₄ are independently a hydrogen or group —C(O)—NR₆R₇, R₆ and R₇ being the same or different for R₃ and R₄ and represent, independently, a hydrogen, or a saturated or unsaturated branched or linear polyalkylamine, wherein one or more amine units in said polyalkylamine may be a quaternary ammonium; provided that R₃ and R₄ are not simultaneously a hydrogen; or R₃ and R₄ form together with the oxygen atoms to which they are bound a heterocyclic ring comprising —C(O)—NR₉—[R₈—NR₉]_(m)—C(O)—, R₈ represents a saturated or unsaturated C₁-C₄ alkyl and R₉ represents a hydrogen or a polyalkylamine of the formula —[R₈—NR₉]_(n)—, wherein said R₉ or each alkylamine unit R₈NR₉ may be the same or different in said polyalkylamine; and n and m, represent independently an integer from 1 to 10; W represents a group selected from —CH═CH—, —CH₂—CH(OH)— or —CH₂—CH₂—.
 13. The method of claim 12, wherein R₁ represents a —C(O)R₅ group; R₅ represents a C₁₂-C₁₈ linear or branched alkyl or alkenyl; W represents —CH═CH—; R₂ represents a C₁₂-C₁₈ linear or branched alkyl or alkenyl; R₃ and R₄ represent, independently, a hydrogen or a group C(O)—NR₆R₇, wherein said R₃ and R₄ are not simultaneously a hydrogen, and R₆ and R₇ represent, independently, a hydrogen or a polyalkylamine having the general formula (II):

wherein R₈ represent a C₁-C₄ alkyl; R₉ represents a hydrogen or a polyalkylamine branch of formula (II), said R₈ and R₉ may be the same or different for each alkylamine unit, —R₈NR₉—, in the polyalkylamine of formula (II); and n represents an integer from 3 to
 6. 14. The method of claim 13, wherein R₃ or R₄ is a hydrogen atom.
 15. The method of claim 13, wherein both R₃ and R₄ represent the same or a different polyalkylamine.
 16. The method of claim 13, wherein R₁ represents a C(O)R₅ group; R₅ represents a C₁₂-C₁₈ linear or branched alkyl or alkenyl; W represents —CH═CH—; R₂ represents a C₁₂-C₁₈ linear or branched alkyl or alkenyl; R₃ and R₄ form together with the oxygen atoms to which they are bonded a heterocyclic ring comprising —C(O)—[NH—R₈]_(n)—NH—C(O)—, wherein R₈ represents a C₁-C₄ alkyl, wherein for each alkylamine unit having the formula —NH—R₈—, said R₈ may be the same or different; and n represents an integer from 3 to
 6. 17. The method of claim 13, wherein said R₈ is a C₃-C₄ alkyl.
 18. The method of claim 1, comprising intranasal or parenteral administration of said combination of sphingoid-polyalkylamine conjugate and said biologically active molecule.
 19. The method of claim 1, comprising intranasal or intramuscular administration of a combination of said sphingoid-polyalkylamine conjugate with said biologically active molecule, wherein: (i) when said infection is caused by HBV, said biologically active molecule is an HBV antigen; (ii) when said infection is caused by avian influenza virus, said biologically active molecule is an AIV antigen; (iii) when said infection is caused by the bacterium Bacillus anthracis, said biologically active molecule is an antigen of said bacterium; or (iv) when said infection is caused by the bacterium Streptococcus pneumoniae said biologically active molecule is an antigen of said bacterium.
 20. The method of claim 19, wherein: (i) said HBV antigen is an HBV surface particle (HBsAg); (ii) said AIV antigen is an inactivated purified whole avian influenza virus; (iii) said bacterium antigen is the protective antigen (PA) moiety of anthrax toxin; or (iv) said Streptococcus pneumoniae antigen is a polysaccharide or protein conjugate of an antigen selected from pneumococcal surface protein A (PspA), pneumococcal adherence and virulence factor A (PavA), pneumococcal glutamyl-tRNA synthetase, pneumolysin, cholin binding protein A (CbpA), pneumococcal surface adhesion A (PsaA).
 21. The method of claim 13, comprising intranasal or intramuscular administration of said N-palmitoyl D-erythro sphingosyl carbamoyl-spermine together with said biologically active molecule.
 22. A vaccine comprising a combination of a sphingoid-polyalkylamine conjugate and an amount of a biologically active molecule, the amount of said biologically active molecule, when combined with said sphingoid-polyalkylamine conjugate, being effective to stimulate or enhance an immune response of a subject to provide protection against an infection caused by an agent selected from the group consisting of HBV, AIV, the bacterium Bacillus anthracis and the bacterium Streptococcus pneumoniae.
 23. The vaccine of claim 22, further comprising an immunostimulating agent.
 24. The vaccine of claim 22, wherein said sphingoid-polyalkylamine conjugate comprises a sphingoid backbone carrying, via a carbamoyl linkage at lest one polyalkylamine chain.
 25. The vaccine of claim 22, wherein said sphingoid backbone is selected from ceramide, dihydroceramide, phytoceramide, dihydrophytoceramide, ceramine, dihydroceramine, phytoceramine, dihydrophytoceramine.
 26. The vaccine of claim 22, wherein said sphingoid is ceramide and said polyalkylamine chain is selected from spermine, spermidine or a polyalkylamine analog of spermine or spermidine.
 27. The vaccine of claim 22, wherein said sphingoid-polyalkylamine conjugate comprises N-palmitoyl-D-erythro-sphingosyl carbamoyl-spermine (CCS).
 28. The vaccine of claim 22, wherein: (i) when said infection is caused by HBV, said biologically active molecule is an HBsAg particle; (ii) when said infection is caused by avian influenza virus, said biologically active molecule is an inactivated purified whole avian influenza virus; (iii) when said infection is caused by the bacterium Bacillus anthracis, said biologically active molecule is a protective antigen (PA) moiety of anthrax toxin or (iv) said Streptococcus pneumoniae antigen is a polysaccharide or protein conjugate of an antigen selected from pneumococcal surface protein A (PspA), pneumococcal adherence and virulence factor A (PavA), pneumococcal glutamyl-tRNA synthetase, pneumolysin, cholin binding protein A (CbpA), pneumococcal surface adhesion A (PsaA).
 29. A vaccine comprising a combination of N-palmitoyl D-erythro sphingosyl carbamoyl-spermine (CCS), being a single isomer of CCS or mixture of CCS isomers, with a biologically active molecule selected from the group consisting of HBsAg particle; an inactivated purified whole avian influenza virus comprising haemagglutinin (H5) antigen; the protective antigen (PA) moiety of anthrax toxin and pneumococcal surface protein A (PspA), or pneumococcal surface adhesion A (PsaA).
 30. The vaccine of claim 22, wherein said sphingoid-polyalkylamine conjugate has the following formula (I):

wherein R₁ represents a hydrogen, a branched or linear alkyl, saturated or unsaturated cycloalkyl, aryl, hydroxyalkyl, alkylamine, or a group —C(O)R₅; R₂ and R₅ represent, independently, a branched or linear C₁₀-C₂₄ alkyl, hydroxyalkyl, alkenyl, saturated or unsaturated cycloalkyl, aryl or polyenyl groups; R₃ and R₄ are independently a hydrogen or a group —C(O)—NR₆R₇, R₆ and R₇ being the same or different for R₃ and R₄ and represent, independently, a hydrogen, or a saturated or unsaturated branched or linear polyalkylamine, wherein one or more amine units in said polyalkylamine may be a quaternary ammonium; wherein said R₄ and R₃ are not simultaneously a hydrogen; or R₃ and R₄ form together with the oxygen atoms to which they are bound a heterocyclic ring comprising —C(O)—NR₉—[R₈—NR₉]_(m)—C(O)—, R₈ represents a saturated or unsaturated C₁-C₄ alkyl and R₉ represents a hydrogen or a polyalkylamine of the formula —[R₈—NR₉],-, wherein said R₉ or each alkylamine unit R₈NR₉ may be the same or different in said polyalkylamine; and n and m, represent independently an integer from 1 to 10; W represents a group selected from —CH═CH—, —CH₂—CH(OH)— or —CH₂—CH₂—.
 31. A complex comprising a sphingoid-polyalkylamine conjugate and a biologically active molecule, the complex being capable of enhancing or stimulating an immune response of a subject to provide protection against an infection caused by an agent selected from HBV, AIV, the bacterium Bacillus anthracis or the bacterium Streptococcus pneumoniae.
 32. The complex of claim 31, comprising N-palmitoyl D-erythro sphingosyl carbamoyl spermine (CCS) associated with said biologically active molecule.
 33. A method for the treatment or prevention of a disease or disorder comprising administering to a subject in need of the treatment a composition comprising a biologically active molecule and a sphingoid-polyalkylamine conjugate having the general formula (I′):

wherein R₁ represents a hydrogen, a branched or linear alkyl, saturated or unsaturated cycloalkyl, aryl, hydroxyalkyl, alkylamine, or a group —C(O)R₅; R₂ and R₅ represent, independently, a branched or linear C₁₀-C₂₄ alkyl, hydroxyalkyl, alkenyl, saturated or unsaturated cycloalkyl, aryl or polyenyl groups; R₃ is a group —C(O)—NR₆R₇, R₆ and R₇ represent, independently, a hydrogen, or a saturated or unsaturated branched or linear polyalkylamine, wherein one or more amine units in said polyalkylamine may be a quaternary ammonium; W represents a group selected from —CH═CH—, —CH₂—CH(OH)— or —CH₂—CH₂—.
 34. The method of claim 33, for stimulating or enhancing an immune response of a subject to provide protection against an infection.
 35. The method of claim 33, wherein said sphingoid-polyalkylamine conjugate is N-palmitoyl D-erythro sphingosyl-3-carbamoyl spermine.
 36. The method of claim 33, wherein said composition comprises a mixture of a first sphingoid-polyalkylamine conjugate of said formula (I′) and a second sphingoid-polyalkylamine conjugate of the following formula (I):

wherein R₁ represents a hydrogen, a branched or linear alkyl, saturated or unsaturated cycloalkyl, aryl, hydroxyalkyl, alkylamine, or a group —C(O)R₅; R₂ and R₅ represent, independently, a branched or linear C₁₀-C₂₄ alkyl, hydroxyalkyl, alkenyl, saturated or unsaturated cycloalkyl, aryl or polyenyl groups; R₃ and R₄ are independently a group —C(O)—NR₆R₇, R₆ and R₇ being the same or different for R₃ and R₄ and represent, independently, a hydrogen, or a saturated or unsaturated branched or linear polyalkylamine, wherein one or more amine units in said polyalkylamine may be a quaternary ammonium; or R₃ is a hydrogen; or R₃ and R₄ form together with the oxygen atoms to which they are bound a heterocyclic ring comprising —C(O)—NR₉—[R₈—NR₉]_(m)—C(O)—, R₈ represents a saturated or unsaturated C₁-C₄ alkyl and R₉ represents a hydrogen or a polyalkylamine of the formula —[R₈—NR₉]_(n)—, wherein said R₉ or each alkylamine unit R₈NR₉ may be the same or different in said polyalkylamine; and n and m represent independently an integer from 1 to 10; W represents a group selected from —CH═CH—, —CH₂—CH(OH)— or —CH₂—CH₂—.
 37. The method of claim 36, wherein said mixture comprises a first sphingoid-polyalkylamine conjugate being N-palmitoyl-D-erythro-sphingosyl-3-carbamoyl spermine and a second sphingoid-polyalkylamine conjugate being N-palmitoyl-D-erythro-sphingosyl-1-carbamoyl spermine. 